On the sequential determinants of calpain cleavage

J Biol Chem. 2004 May 14;279(20):20775-85. doi: 10.1074/jbc.M313873200. Epub 2004 Feb 26.


The structural clues of substrate recognition by calpain are incompletely understood. In this study, 106 cleavage sites in substrate proteins compiled from the literature have been analyzed to dissect the signal for calpain cleavage and also to enable the design of an ideal calpain substrate and interfere with calpain action via site-directed mutagenesis. In general, our data underline the importance of the primary structure of the substrate around the scissile bond in the recognition process. Significant amino acid preferences were found to extend over 11 residues around the scissile bond, from P(4) to P(7)'. In compliance with earlier data, preferred residues in the P(2) position are Leu, Thr, and Val, and in P(1) Lys, Tyr, and Arg. In position P(1) ', small hydrophilic residues, Ser and to a lesser extent Thr and Ala, occur most often. Pro dominates the region flanking the P(2)-P(1)' segment, i.e. positions P(3) and P(2)'-P(4)'; most notable is its occurrence 5.59 times above chance in P(3)'. Intriguingly, the segment C-terminal to the cleavage site resembles the consensus inhibitory region of calpastatin, the specific inhibitor of the enzyme. Further, the position of the scissile bond correlates with certain sequential attributes, such as secondary structure and PEST score, which, along with the amino acid preferences, suggests that calpain cleaves within rather disordered segments of proteins. The amino acid preferences were confirmed by site-directed mutagenesis of the autolysis sites of Drosophila calpain B; when amino acids at key positions were changed to less preferred ones, autolytic cleavage shifted to other, adjacent sites. Based on these preferences, a new fluorogenic calpain substrate, DABCYLTPLKSPPPSPR-EDANS, was designed and synthesized. In the case of micro- and m-calpain, this substrate is kinetically superior to commercially available ones, and it can be used for the in vivo assessment of the activity of these ubiquitous mammalian calpains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Autolysis
  • Base Sequence
  • Calpain / chemistry*
  • Calpain / metabolism*
  • Cysteine Endopeptidases / metabolism
  • DNA Primers
  • Isoenzymes / chemistry
  • Isoenzymes / metabolism
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Substrate Specificity


  • DNA Primers
  • Isoenzymes
  • Peptide Fragments
  • Calpain
  • Cysteine Endopeptidases