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Accepted Manuscript
Research Article
Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells
Koichi Ojima, Shoji Hata, Fumiko Shinkai-Ouchi, Mika Oe, Susumu Muroya, Hiroyuki Sorimachi, Yasuko Ono
Biology Open 2020 : bio.048975 doi: 10.1242/bio.048975 Published 14 August 2020
Koichi Ojima
1Muscle Biology Research Unit, Division of Animal Products Research, Institute of Livestock and Grassland Science, NARO, Tsukuba, Japan
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  • ORCID record for Koichi Ojima
  • For correspondence: koojima@affrc.go.jp
Shoji Hata
2Calpain Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
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Fumiko Shinkai-Ouchi
2Calpain Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
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Mika Oe
1Muscle Biology Research Unit, Division of Animal Products Research, Institute of Livestock and Grassland Science, NARO, Tsukuba, Japan
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Susumu Muroya
1Muscle Biology Research Unit, Division of Animal Products Research, Institute of Livestock and Grassland Science, NARO, Tsukuba, Japan
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Hiroyuki Sorimachi
2Calpain Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
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Yasuko Ono
2Calpain Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
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Abstract

Calpain-3 (CAPN3) is a muscle specific type of calpain whose protease activity is triggered by Ca2+. Here, we developed CAPN3 sensor probes (SPs) to detect activated-CAPN3 using a fluorescence/Förster resonance energy transfer (FRET) technique. In our SPs, partial amino acid sequence of calpastatin, endogenous CAPN inhibitor but CAPN3 substrate, is inserted between two different fluorescence proteins which cause FRET. Biochemical and spectral studies revealed that CAPN3 cleaved SPs and changed emission wavelengths of SPs. Importantly, SPs were scarcely cleaved by CAPN1 and CAPN2. Furthermore, our SP successfully captured the activation of endogenous CAPN3 in living myotubes treated with ouabain. Our SPs would become a promising tool to detect the dynamics of CAPN3 protease activity in living cells.

  • Received October 24, 2019.
  • Accepted July 28, 2020.
  • © 2020. Published by The Company of Biologists Ltd
http://creativecommons.org/licenses/by/4.0

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Keywords

  • Calpain
  • Calpain-3
  • Skeletal muscle
  • Calpainopathy
  • Limb-girdle muscular dystrophy type 2A
  • Proteolysis

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Accepted Manuscript
Research Article
Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells
Koichi Ojima, Shoji Hata, Fumiko Shinkai-Ouchi, Mika Oe, Susumu Muroya, Hiroyuki Sorimachi, Yasuko Ono
Biology Open 2020 : bio.048975 doi: 10.1242/bio.048975 Published 14 August 2020
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Accepted Manuscript
Research Article
Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells
Koichi Ojima, Shoji Hata, Fumiko Shinkai-Ouchi, Mika Oe, Susumu Muroya, Hiroyuki Sorimachi, Yasuko Ono
Biology Open 2020 : bio.048975 doi: 10.1242/bio.048975 Published 14 August 2020

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