Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells.

Koichi Ojima,Susumu Muroya,Yasuko Ono,Fumiko Shinkai-Ouchi,Mika Oe,Hiroyuki Sorimachi,Shoji Hata

doi:10.1242/bio.048975

Abstract

ABSTRACTCalpain-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 that 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.

Highlights

Calpain (CAPN) is an intracellular non-lysosomal Ca2+-requiring cysteine protease (EC 3.4.22.17; Clan CA, family C02), which is expressed in cells of humans and other organisms in a tissue specific or ubiquitous fashion (Goll et al, 2003)
Design of sensor probes (SPs) cleaved by CAPN3 To capture signals of activated-CAPN3 in living cells, we developed Fö rster resonance energy transfer (FRET)-based SPs, which consisted of enhanced cyan fluorescence protein (CFP), partial CAST sequence and mutated yellow fluorescence protein (Venus)
The ratio of CFP/Venus was relatively constant in cultured Capn3C129S/C129S muscle cells (Fig. 4D). These results demonstrated that SP#3 was cleaved by endogenous CAPN3 activated in cultured muscle cells that were treated with ouabain

Summary

Introduction

Calpain (CAPN) is an intracellular non-lysosomal Ca2+-requiring cysteine protease (EC 3.4.22.17; Clan CA, family C02), which is expressed in cells of humans and other organisms in a tissue specific or ubiquitous fashion (Goll et al, 2003). In contrast to other intracellular proteases such as caspases that proteolyze their substrates at relatively conservative peptide sequences in their substrates (Julien and Wells, 2017), CAPNs’ universal consensus amino acid sequences to be cleaved by CAPNs have not been identified in their substrates, i.e. CAPNs’ substrate preference motif is still ambiguous (Shinkai-Ouchi et al, 2016). It is likely that CAPNs recognize a connecting region between structural domains rather than consensus amino acid residues in its substrate as a cleavage site (Tompa et al, 2004). As CAPN regulates cellular process through limited cleavage of substrates, CAPN is recognized as a modulator protease

Methods

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Conclusion