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木原孝洋

徳島大学

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2025年11月7日更新

職名: 特任准教授
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学位: 博士 / 博士(理学)
職歴・経歴: 2025/10: 徳島大学特任准教授, フォトニクス健康フロンティア研究院

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著書

木原孝洋 :
動物用再生医療等製品の開発に必要な試験法等に関する自主基準第1版(2022年), --- 第2章有効性に係わる細胞の品質管理 ---,
一般社団法人動物再生医療推進協議会, 相模原, 2022年9月. 桑原大幹, 木原孝洋, 田中純子, 南野直人 :
ポストゲノム・マススペクトロメトリー(生化学者のための生体高分子解析), --- ペプチドーム解析 ---,
株式会社化学同人, 京都, 2003年7月. 木原孝洋, 桑原大幹, 田中純子, 南野直人 :
プロテオミクス -方法とその病態解析への応用-, 現代化学増刊42, --- ペプチドーム解析 ---,
株式会社東京化学同人, 東京, 2002年.

論文

Susanne Frykman, Mitsuhiro Inoue, Atsushi Ikeda, Yasuhiro Teranishi, Takahiro Kihara, L. Jolanta Lundgren, G. Natsuko Yamamoto, Nenad Bogdanovic, Bengt Winblad, Sophia Schedin-Weiss and O. Lars Tjernberg :
Maturation and processing of the amyloid precursor protein is regulated by the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2),
Biochemical and Biophysical Research Communications, 483, 1, 352-358, 2016.

(要約): The toxic amyloid β-peptide (Aβ) is a key player in Alzheimer Disease (AD) pathogenesis and selective inhibition of the production of this peptide is sought for. Aβ is produced by the sequential cleavage of the Aβ precursor protein (APP) by β-secretase (to yield APP-C-terminal fragment β (APP-CTFβ) and soluble APPβ (sAPPβ)) and γ-secretase (to yield Aβ). We reasoned that proteins that associate with γ-secretase are likely to regulate Aβ production and to be targets of pharmaceutical interventions and therefore performed a pull-down assay to screen for such proteins in rat brain. Interestingly, one of the purified proteins was potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2), which has been shown to be involved in epilepsy. We found that silencing of HCN2 resulted in decreased secreted Aβ levels. To further investigate the mechanism behind this reduction, we also determined the levels of full-length APP, sAPP and APP-CTF species after silencing of HCN2. A marked reduction in sAPP and APP-CTF, as well as glycosylated APP levels was detected. Decreased Aβ, sAPP and APP-CTF levels were also detected after treatment with the HCN2 inhibitor ZD7288. These results indicate that the effect on Aβ levels after HCN2 silencing or inhibition is due to altered APP maturation or processing by β-secretase rather than a direct effect on γ-secretase. However, HCN2 and γ-secretase were found to be in close proximity, as evident by proximity ligation assay and immunoprecipitation. In summary, our results indicate that silencing or inhibition of HCN2 affects APP processing and thereby could serve as a potential treatment strategy.
(キーワード): Alzheimer disease / Amyloid beta-peptide / Amyloid precursor protein / Glycosylation / Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2
(出版サイトへのリンク): ● Publication site (DOI): 10.1016/j.bbrc.2016.12.140
(文献検索サイトへのリンク): ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 28017718; ● Summary page in Scopus @ Elsevier: 2-s2.0-85009262063

(DOI: 10.1016/j.bbrc.2016.12.140, PubMed: 28017718, Elsevier: Scopus) Yasuhiro Teranishi, Mitsuhiro Inoue, Goto Natsuko Yamamoto, Takahiro Kihara, Birgitta Wiehager, Taizo Ishikawa, Bengt Winblad, Sophia Schedin-Weiss, Susanne Frykman and O. Lars Tjernberg :
Proton myo-inositol cotransporter is a novel γ-secretase associated protein that regulates Aβ production without affecting Notch cleavage,
The FEBS Journal, 282, 17, 3438-3451, 2015.

(要約): γ-Secretase is a transmembrane protease complex that is responsible for the processing of a multitude of type 1 transmembrane proteins, including the amyloid precursor protein and Notch. γ-Secretase processing of amyloid precursor protein results in the release of the amyloid β-peptide (Aβ), which is involved in the pathogenesis in Alzheimer's disease. Processing of Notch leads to the release of its intracellular domain, which is important for cell development. γ-Secretase associated proteins (GSAPs) could be of importance for substrate selection, and we have previously shown that affinity purification of γ-secretase in combination with mass spectrometry can be used for finding such proteins. In the present study, we used this methodology to screen for novel GSAPs from human brain, and studied their effect on Aβ production in a comprehensive gene knockdown approach. Silencing of probable phospholipid-transporting ATPase IIA, brain-derived neurotrophic factor/neurotrophin-3 growth factor receptor precursor and proton myo-inositol cotransporter (SLC2A13) showed a clear reduction of Aβ and these proteins were selected for further studies on Aβ production and Notch cleavage using small interfering RNA-mediated gene silencing, as well as an overexpression approach. Silencing of these reduced Aβ secretion in a small interfering RNA dose-dependent manner. Interestingly, SLC2A13 had a lower effect on Notch processing. Furthermore, overexpression of SLC2A13 increased Aβ40 generation. Finally, the interaction between γ-secretase and SLC2A13 was confirmed using immunoprecipitation and a proximity ligation assay. In summary, SLC2A13 was identified as a novel GSAP that regulates Aβ production without affecting Notch cleavage. We suggest that SLC2A13 could be a target for Aβ lowering therapy aimed at treating Alzheimer's disease.
(キーワード): Alzheimer's disease / amyloid β-peptide / Notch / proton myo-inositol cotransporter / γ-secretase
(出版サイトへのリンク): ● Publication site (DOI): 10.1111/febs.13353
(文献検索サイトへのリンク): ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 26094765; ● Summary page in Scopus @ Elsevier: 2-s2.0-84940721622

(DOI: 10.1111/febs.13353, PubMed: 26094765, Elsevier: Scopus) Yasuhiro Teranishi, Yeun Ji Hur, Jijuan Gucci Gu, Takahiro Kihara, Taizo Ishikawa, Takeshi Nishimura, Bengt Winblad, Homira Behbahani, Masood Kamali-Moghaddam, Susanne Frykman and O. Lars Tjernberg :
Erlin-2 is associated with active γ-secretase in brain and affects amyloid β-peptide production,
Biochemical and Biophysical Research Communications, 424, 3, 476-481, 2012.

(要約): The transmembrane protease complex γ-secretase is responsible for the generation of the neurotoxic amyloid β-peptide (Aβ) from its precursor (APP). Aβ has a causative role in Alzheimer disease, and thus, γ-secretase is a therapeutic target. However, since there are more than 70 γ-secretase substrates besides APP, selective inhibition of APP processing is required. Recent data indicates the existence of several γ-secretase associated proteins (GSAPs) that affect the selection and processing of substrates. Here, we use a γ-secretase inhibitor for affinity purification of γ-secretase and associated proteins from microsomes and detergent resistant membranes (DRMs) prepared from rat or human brain. By tandem mass spectrometry we identified a novel brain GSAP; erlin-2. This protein was recently reported to reside in DRMs in the ER. A proximity ligation assay, as well as co-immunoprecipitation, confirmed the association of erlin-2 with γ-secretase. We found that a higher proportion of erlin-2 was associated with γ-secretase in DRMs than in soluble membranes. siRNA experiments indicated that reduced levels of erlin-2 resulted in a decreased Aβ production, whereas the effect on Notch processing was limited. In summary, we have found a novel brain GSAP, erlin-2, that resides in DRMs and affects Aβ production.
(キーワード): γ-Secretase / Affinity purification / Alzheimer disease / Amyloid β-peptide / Detergent resistant membranes / Erlin-2
(出版サイトへのリンク): ● Publication site (DOI): 10.1016/j.bbrc.2012.06.137
(文献検索サイトへのリンク): ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 22771797; ● Summary page in Scopus @ Elsevier: 2-s2.0-84864740457

(DOI: 10.1016/j.bbrc.2012.06.137, PubMed: 22771797, Elsevier: Scopus) Yeun Ji Hur, Yasuhiro Teranishi, Takahiro Kihara, Goto Natsuko Yamamoto, Mitsuhiro Inoue, Waltteri Hosia, Masakazu Hashimoto, Bengt Winblad, Susanne Frykman and O. Lars Tjernberg :
Identification of novel γ-secretase-associated proteins in detergent-resistant membranes from brain,
The Journal of Biological Chemistry, 287, 15, 11991-12005, 2012.

(要約): In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.
(キーワード): Amino Acid Sequence / Amyloid Precursor Protein Secretases / Amyloid beta-Peptides / Animals / 脳 (brain) / Cell Adhesion Molecules, Neuronal / Chromatography, Affinity / Gene Knockdown Techniques / HEK293 Cells / Humans / 男性 (male) / Membrane Glycoproteins / Membrane Microdomains / Membrane Proteins / Molecular Sequence Data / Peptide Fragments / Presenilin-1 / Protein Binding / Rats / Rats, Sprague-Dawley / Receptors, Notch / Syntaxin 1 / Tandem Mass Spectrometry / Voltage-Dependent Anion Channel 1
(出版サイトへのリンク): ● Publication site (DOI): 10.1074/jbc.M111.246074
(文献検索サイトへのリンク): ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 22315232; ● Summary page in Scopus @ Elsevier: 2-s2.0-84859488864

(DOI: 10.1074/jbc.M111.246074, PubMed: 22315232, Elsevier: Scopus) Yoshihiro Oyamada, Ichi Jun Yamagishi, Takahiro Kihara, Hiroaki Yoshida, Masaaki Wachi and Hideaki Ito :
Mechanism of inhibition of DNA gyrase by ES-1273, a novel DNA gyrase inhibitor,
Microbiology and Immunology, 51, 10, 977-984, 2007.

(要約): We investigated the mode of action of ES-1273, a novel DNA gyrase inhibitor obtained by optimization of ES-0615, which was found by screening our chemical library using anucleate cell blue assay. ES-1273 exhibited the same antibacterial activity against S. aureus strains with amino acid change(s) conferring quinolone- and coumarin-resistance as that against a susceptible strain. In addition, ES-1273 inhibited DNA gyrase supercoiling activity, but not ATPase activity of the GyrB subunit of DNA gyrase. Moreover, ES-1273 did not induce cleavable complex. These findings demonstrate that the mechanism by which ES-1273 inhibits DNA gyrase is different from that of the quinolones or the coumarins. Preincubation of DNA gyrase and substrate DNA prevented inhibition of DNA gyrase supercoiling activity by ES-1273. ES-1273 antagonized quinolone-induced cleavage. In electrophoretic mobility shift assay, no band representing DNA gyrase-DNA complex was observed in the presence of ES-1273. Taken together, these results indicate that ES-1273 prevents DNA from binding to DNA gyrase. Furthermore, our results from surface plasmon resonance experiments strongly suggest that ES-1273 interacts with DNA. Therefore, the interaction between ES-1273 and DNA prevents DNA from binding to DNA gyrase, resulting in inhibition of DNA gyrase supercoiling. Interestingly, we also found that ES-1273 inhibits topoisomerase IV and human topoisomerase IIalpha, but not human topoisomerase I. These findings indicate that ES-1273 is a type II topoisomerase specific inhibitor.
(キーワード): A gyrase inhibitor / DNA gyrase / Topoisomerase IV
(出版サイトへのリンク): ● Publication site (DOI): 10.1111/j.1348-0421.2007.tb03994.x
(文献検索サイトへのリンク): ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 17951987; ● Summary page in Scopus @ Elsevier: 2-s2.0-35448950270

(DOI: 10.1111/j.1348-0421.2007.tb03994.x, PubMed: 17951987, Elsevier: Scopus) 南野直人, 桑原大幹, 松井泰子, 木原孝洋, 磯山-田中純子 :
ペプチドーム研究の最前線,
化学と生物, 42, 3, 162-169, 2004年.

(出版サイトへのリンク): ● Publication site (DOI): 10.1271/kagakutoseibutsu1962.42.162
(文献検索サイトへのリンク): ● CiNii @ 国立情報学研究所 (CRID): 1390001204200425856; ● Search Scopus @ Elsevier (DOI): 10.1271/kagakutoseibutsu1962.42.162

(DOI: 10.1271/kagakutoseibutsu1962.42.162, CiNii: 1390001204200425856) Naoto Minamino, Junko Tanaka, Hiromiki Kuwahara, Takahiro Kihara, Yoshinori Satomi, Masami Matsubae and Toshifumi Takao :
Determination of endogenous peptides in the porcine brain: Possible construction of Peptidome, a fact database for endogenous peptides,
Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 792, 1, 33-48, 2003.

(要約): Peptides play crucial roles in many physiological events. However, a database for endogenous peptides has not yet been developed, because the peptides are easily degraded by proteolytic enzymes during extraction and purification. In this study, we demonstrated that the data for endogenous peptides could be collected by minimizing the proteolytic degradation. We separated porcine brain peptides into 5250 fractions by 2-dimensional chromatography (first ion-exchange and second reversed-phase high-performance liquid chromatography), and 75 fractions of average peptide contents were analyzed in detail by mass spectrometers and a protein sequencer. Based on the analysis data obtained in this study, more than 10000 peptides were deduced to be detected, and more than 1000 peptides to be identified starting from 2 g of brain tissue. Thus, we deduce that it is possible to construct a database for endogenous peptides starting from a gram level of tissue by using 2-dimensional high-performance liquid chromatography coupled with a mass spectrometer.
(キーワード): Peptides, endogenous / Peptidome
(出版サイトへのリンク): ● Publication site (DOI): 10.1016/S1570-0232(03)00280-0
(文献検索サイトへのリンク): ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 12828995; ● Summary page in Scopus @ Elsevier: 2-s2.0-0038798804

(DOI: 10.1016/S1570-0232(03)00280-0, PubMed: 12828995, Elsevier: Scopus) Ryuichi Ohkura, Atsushi Kimura, Takahiro Kihara, Katsueki Ogiwara and Takayuki Takahashi :
Expression of bradykinin B2 receptor in the mouse ovary,
Zoological Science, 20, 7, 847-854, 2003.

(キーワード): Bradykinin B2 receptor / Expression / Mouse / 卵巣 (ovary)
(出版サイトへのリンク): ● Publication site (DOI): 10.2108/zsj.20.847
(文献検索サイトへのリンク): ● Summary page in Scopus @ Elsevier: 2-s2.0-0141484640

(DOI: 10.2108/zsj.20.847, Elsevier: Scopus) 磯山(田中) 純子, 桑原大幹, 木原孝洋, 南野直人 :
生体内ペプチドのファクトデータベース「ペプチドーム」と蛋白質代謝のメタボローム,
炎症と免疫, 11, 657-664, 2003年. 桑原大幹, 木原孝洋, 田中純子, 南野直人 :
ペプチドームプロジェクト -生体内ペプチドのファクトデータベースの構築-,
生物物理, 42, 3, 122-126, 2002年.

(出版サイトへのリンク): ● Publication site (DOI): 10.2142/biophys.42.122
(文献検索サイトへのリンク): ● Search Scopus @ Elsevier (DOI): 10.2142/biophys.42.122

(DOI: 10.2142/biophys.42.122) A. Kimura, Takahiro Kihara, R. Ohkura, K. Ogiwara and T. Takahashi :
Localization of bradykinin B2 receptor in the follicles of porcine ovary and increased expression of matrix metalloproteinase-3 and -20 in cultured granulosa cells by bradykinin treatment,
Biology of Reproduction, 65, 5, 1462-1470, 2001.

(要約): We have recently shown that not only bradykinin, but also all components for the production of bradykinin, can be detected within the follicle of porcine ovaries. To elucidate the relevance of the intrafollicular bradykinin-producing system to its physiological role, we investigated the distribution of bradykinin receptor (B(2)R) mRNA and the protein in porcine ovaries. A cDNA encoding porcine B(2)R was first cloned from a porcine uterus cDNA library. The receptor mRNA was scarcely detected in the ovary by Northern blot analysis. Polymerase chain reaction analysis with total RNAs isolated from the ovary and from granulosa cells of small and large follicles demonstrated the ovarian expression of B(2)R mRNA. The B(2)R protein was detected by Western blot analysis in extracts of isolated granulosa cells. In situ hybridization of B(2)R mRNA and immunohistochemical analysis of the protein revealed that the receptor is expressed in the theca and granulosa cells of all growing follicles. The effect of bradykinin on the expression of some matrix metalloproteinase (MMP) genes was examined using isolated granulosa cells. Bradykinin treatment induced MMP-3 and MMP-20 gene expression to an extreme degree. The expression of MT1-MMP was also affected by bradykinin treatment. These results suggest that MMPs play a role in follicle rupture during ovulation. The present study provides new information regarding the mechanisms of bradykinin-induced ovulation in porcine ovaries.
(キーワード): Follicle / Granulosa cells / 卵巣 (ovary) / Ovulation / Theca cells
(出版サイトへのリンク): ● Publication site (DOI): 10.1095/biolreprod65.5.1462
(文献検索サイトへのリンク): ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 11673263; ● Summary page in Scopus @ Elsevier: 2-s2.0-0034761391

(DOI: 10.1095/biolreprod65.5.1462, PubMed: 11673263, Elsevier: Scopus) Atsushi Kimura, Takahiro Kihara, Hikari Okimura, Takashi Hamabata, Junji Ohnishi, Akihiko Moriyama, Kenji Takahashi and Takayuki Takahashi :
Identification of porcine follipsin as plasma kallikrein, and its possible involvement in the production of bradykinin within the follicles of porcine ovaries,
Molecular Reproduction and Development, 57, 1, 79-87, 2000.

(要約): To determine the identity of porcine follipsin, a plasma kallikrein cDNA clone was isolated from a porcine liver cDNA library. The clone encoded a protein of 643 amino acids, exhibiting identities 79.7, 72. 9, and 74.4% homologous to human, rat, and mouse plasma prekallikrein, respectively. The amino acid sequences of four internal peptides isolated from the tryptic digest of follipsin were all found in the deduced sequence. Authentic plasma kallikrein was purified from porcine plasma and compared directly with follipsin. Actions on synthetic substrates and behaviors with proteinase inhibitors were indistinguishable between these two enzymes. The cDNA was expressed in COS-7 cells and the recombinant protein was prepared from the culture medium of these cells. No active enzyme could be obtained, but the expressed protein was reacted with anti-porcine plasma kallikrein antibody. The mRNA was detected only in the liver in northern blot analysis. RT-PCR analysis of RNAs revealed that porcine testis, in addition to the liver, expressed the corresponding mRNA. In the ovary, plasma kallikrein was detected as a main band of the active form (Mr = 85,000) and the band of the minor inactive precursor form (Mr = 80,000), respectively. In contrast, the liver extract contained only the precursor form. Incubation of high molecular weight kininogen with follicular fluid plasma kallikrein resulted in an increased production of bradykinin. Further, the fresh fluid of large-sized follicles of porcine ovaries was found to contain this peptide hormone at a detectable level. These results indicate that porcine follipsin is plasma kallikrein, and that the enzyme may be involved in the production of bradykinin within ovarian follicles.
(キーワード): Bradykinin / Follicle / Follicular fluid / Pig ovary / Plasma kallikrein
(出版サイトへのリンク): ● Publication site (DOI): 10.1002/1098-2795(200009)57:1<79::AID-MRD11>3.0.CO;2-E
(文献検索サイトへのリンク): ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 10954859; ● Summary page in Scopus @ Elsevier: 2-s2.0-0033871138

(DOI: 10.1002/1098-2795(200009)57:1<79::AID-MRD11>3.0.CO;2-E, PubMed: 10954859, Elsevier: Scopus) Takahiro Kihara, Atsushi Kimura, Akihiko Moriyama, Iwao Ohkubo and Takayuki Takahashi :
Identification of components of the intrafollicular bradykinin-producing system in the porcine ovary,
Biology of Reproduction, 62, 5, 1160-1167, 2000.

(要約): As a step in elucidating the biological role of plasma kallikrein (PK) present in the follicular fluid of mammalian ovaries, we examined pig ovary fluid to determine its constituent activators and substrates. Using the inactive precursor form of plasma kallikrein (prePK) as a substrate, we purified an enzyme capable of activating this protein. The prePK-activating enzyme was shown to be the active enzyme blood coagulation factor XIIa. We also isolated high molecular weight kininogen (HMW-K) from the same fluid. Incubation of HMW-K with the ovarian follicular fluid PK resulted in the production of the nanopeptide bradykinin (BK). Expression of prePK, blood coagulation factor XII, and HMW-K was examined by Northern blot analysis using ovary and liver poly(A)(+) RNA. All these transcripts were found in the liver, but none were found in the ovary. In addition, it was found that BK levels in the fluid derived from the small follicles were approximately 6 times higher than those from medium and large follicles. These results demonstrate the presence of a BK-producing system in the ovarian follicles and suggest the physiological importance of this peptide hormone in the early stages of follicular development and at ovulation.
(キーワード): Amino Acid Sequence / Animals / Bradykinin / Factor XIIa / 女性 (female) / Follicular Fluid / Kallikreins / Kininogen, High-Molecular-Weight / Molecular Sequence Data / Ovarian Follicle / RNA, Messenger / Swine
(出版サイトへのリンク): ● Publication site (DOI): 10.1095/biolreprod62.5.1160
(文献検索サイトへのリンク): ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 10775162; ● Summary page in Scopus @ Elsevier: 2-s2.0-0034095013

(DOI: 10.1095/biolreprod62.5.1160, PubMed: 10775162, Elsevier: Scopus) Takayuki Takahashi, Hitoshi Matsui, Takahiro Kihara, Atsushi Kimura and Junji Ohnishi :
Identification and partial characterization of a metallopeptidase from porcine ovaries,
The Journal of Experimental Zoology, 281, 6, 574-581, 1998.

(要約): The follicular fluid of porcine ovaries contains an EDTA-sensitive enzyme activity for the synthetic substrate benzyloxycarbonyl-Val-Lys-Met-4-methylcoumaryl-7-amide. To investigate its characteristics and its identification, the enzyme was partially purified by ammonium sulfate fractionation followed by column chromatographies on DEAE-Cellulose and chelating Cellulofine columns. The enzyme activity was strongly inhibited by typical chelators, such as EDTA and o-phenanthroline, but after inhibition by EDTA the activity was completely restored with an appropriate amount of Zn2+ and Co2+ ions. It showed enzyme activity solely for benzyloxycarbonyl-Val-Lys-Met-4-methylcoumaryl-7-amide among the substrates tested. The molecular weight of the enzyme was estimated to be 400,000 by gel filtration. The enzyme activity in the fluid obtained from large follicles of porcine ovaries was significantly higher than that from smaller follicles. It appeared that the granulosa cell extract did not contain the metalloenzyme activity. Similar enzyme activities were detected in follicular fluids from bovine and human ovaries. These results suggest that the present enzyme is distinct from any other metalloendopeptidases thus far reported.
(キーワード): Animals / Cations, Divalent / Cattle / Chelating Agents / Chromatography, Affinity / Chromatography, DEAE-Cellulose / Chromatography, Gel / Edetic Acid / Female / Follicular Fluid / Granulosa Cells / Humans / Kinetics / Metalloendopeptidases / Molecular Weight / Ovary / Protease Inhibitors / Substrate Specificity / Swine
(出版サイトへのリンク): ● Publication site (DOI): 10.1002/(SICI)1097-010X(19980815)281:6<574::AID-JEZ5>3.0.CO;2-K
(文献検索サイトへのリンク): ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 9697322; ● Summary page in Scopus @ Elsevier: 2-s2.0-0032529737

(DOI: 10.1002/(SICI)1097-010X(19980815)281:6<574::AID-JEZ5>3.0.CO;2-K, PubMed: 9697322, Elsevier: Scopus) Takahiro Kihara, Junji Ohnishi, Kuniko Kohyama, Akihiko Moriyama and Takayuki Takahashi :
Identification and activation of profollipsin, a latent precursor form of porcine follipsin,
European Journal of Biochemistry, 245, 2, 392-397, 1997.

(要約): A latent protease has been identified in column fractions obtained during the purification of the porcine ovarian serine protease follipsin. The latent enzyme was readily activated by trypsin treatment. The trypsin-activated enzyme was purified using a benzamidine-Sepharose 6B column and was shown to be composed of two distinct, covalently associated polypeptides with Mr of 45000 and 32000. This polypeptide chain composition, together with its substrate specificity, inhibition profile using various protease inhibitors, cross-reactivity with anti-follipsin antibody, and ability to activate single-chain precursor tissue plasminogen activator, indicated its identity as porcine follipsin. The activation of the enzyme with trypsin was found to occur by the hydrolysis of an internal peptide bond resulting in a two-chain structure. Thus, we conclude that the latent enzyme is the inactive precursor form (profollipsin) of follipsin. The present study also shows that the follicular fluid of porcine ovary contains a profollipsin-activating enzyme activity.
(キーワード): Follipsin / Porcine ovary / Proenzyme activation / Profollipsin
(出版サイトへのリンク): ● Publication site (DOI): 10.1111/j.1432-1033.1997.00392.x
(文献検索サイトへのリンク): ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 9151969; ● Summary page in Scopus @ Elsevier: 2-s2.0-0030951801

(DOI: 10.1111/j.1432-1033.1997.00392.x, PubMed: 9151969, Elsevier: Scopus) Junji Ohnishi, Takahiro Kihara, Takashi Hamabata, Kenji Takahashi and Takayuki Takahashi :
Cleavage specificity of porcine follipsin,
The Journal of Biological Chemistry, 270, 33, 19391-19394, 1995.

(要約): Follipsin purified from the follicular fluid of porcine ovaries was studied for its specificity against various synthetic and peptide substrates. The enzyme cleaved only by an endopeptidase activity at the amide and peptide bonds of Arg-X, indicating strict specificity of the S1 pocket for arginine. The specificity for pocket S2 appears to favor either hydrophobic or basic side chains. A 10-residue peptide containing a portion of the activation site of human tissue plasminogen activator was synthesized and tested with the enzyme. The peptide was cleaved by follipsin at the Arg-Ile bond, as expected from the specificity deduced above. Furthermore, the enzyme successfully converted single-chain precursor tissue plasminogen activator (sctPA) to its active, two-chain form by cleaving the corresponding peptide bond. Comparison of the rates of single-chain precursor tissue plasminogen activator activation and tissue plasminogen activator peptide hydrolysis revealed that the former is a more efficient substrate than the latter.
(キーワード): Amino Acid Sequence / Animals / Enzyme Activation / Female / Hydrolysis / Molecular Sequence Data / Ovary / Recombinant Proteins / Serine Endopeptidases / Substrate Specificity / Swine / Urokinase-Type Plasminogen Activator
(出版サイトへのリンク): ● Publication site (DOI): 10.1074/jbc.270.33.19391
(文献検索サイトへのリンク): ● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 7642619; ● Summary page in Scopus @ Elsevier: 2-s2.0-0029150270

(DOI: 10.1074/jbc.270.33.19391, PubMed: 7642619, Elsevier: Scopus)

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講演・発表: Takahiro Kihara, A Kimura, J Ohnishi and T Takahashi :
A latent precursor form of follipsin and its activating enzyme,
XIII International Congress of Comparative Endcrinology 1997, 1545-1548, 1997. T Takahashi, J Ohnishi, Takahiro Kihara and A Kimura :
Proteinases involved in follicle rupture during ovulation,
XIII International Congress of Comparative Endcrinology 1997, 1463-1468, 1997.

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特許: 上田忠佳, 木原孝洋 : 低温輸送容器及び低温輸送容器への物品の梱包方法, 特願2021-035584, . 木原孝洋, 黒宮明美 : 細胞凝集低減方法および細胞凝集が低減された治療用組成物, 特許第WO 2018/225673 A1号. O.Lars Tjernberg, Susanne Frykman, Ji-Yeun Hur, 寺西康博, 中川博之, 左海順, 木原孝洋 : アミロイドベータペプチドの産生を促進する因子を用いた阻害剤のスクリーニング方法及びそれによって得られる阻害剤, 特許第WO 2010/140694 A1号.
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