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Calpain 1–titin interactions concentrate calpain 1 in the Z‐band edges and in the N2‐line region within the skeletal myofibril
Author(s) -
Raynaud Fabrice,
Fernandez Eric,
Coulis Gerald,
Aubry Laurent,
Vig Xavier,
Bleimling Nathalie,
Gautel Mathias,
Benyamin Yves,
Ouali Ahmed
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04683.x
Subject(s) - calpain , titin , myofibril , immunoelectron microscopy , proteolysis , obscurin , chemistry , protease , skeletal muscle , biophysics , microbiology and biotechnology , biology , anatomy , biochemistry , sarcomere , myocyte , immunohistochemistry , enzyme , immunology
Calpain 1, a ubiquitous calcium‐dependent intracellular protease, was recently found in a tight association with myofibrils in skeletal muscle tissue [Delgado EF, Geesink GH, Marchello JA, Goll DE & Koohmaraie M (2001) J Anim Sci 79 , 2097–2107). Our immunofluorescence and immunoelectron microscopy investigations restrain the protease location at the periphery of the Z‐band and at the midpoint of the I‐band. Furthermore, calpain 1 is found to localize in myofibril fractures, described as proteolysis sites, in postmortem bovine skeletal red muscles, near the calcium deposits located at the N1 and N2 level. This in situ localization of calpain 1 is substantiated by binding assays with two titin regions covering the I‐band region: a native fragment of 150 kDa (identified by mass spectrometry) that includes the N‐terminal Z8–I5 region and the N1‐line region of titin, and an 800 kDa fragment external to the N1 line that bears the PEVK/N2 region. These two titin fragments are shown to tightly bind calpain 1 in the presence of CaCl 2 and E64, a calpain inhibitor. In the absence of E64, they are cleaved by calpain 1. We conclude that titin affords binding sites to calpain 1, which concentrates the protease in the regions restrained by the Z‐band edge and the N1‐line as well as at the N2‐line level, two sarcomeric regions where early postmortem proteolysis is detected.