On the sequential determinants of calpain cleavage

Peter Tompa, Peter Buzder-Lantos, Agnes Tantos, Attila Farkas, András Szilágyi, Z. Bánóczi, F. Hudecz, P. Friedrich

Research output: Contribution to journalArticle

243 Citations (Scopus)

Abstract

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 P4 to P7′. In compliance with earlier data, preferred residues in the P2 position are Leu, Thr, and Val, and in P1 Lys, Tyr, and Arg. In position P1′, small hydrophilic residues, Ser and to a lesser extent Thr and Ala, occur most often. Pro dominates the region flanking the P2-P1′ segment, i.e. positions P3 and P2′-P 4′; most notable is its occurrence 5.59 times above chance in P3′. 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, DABCYL-TPLKSPPPSPR-EDANS, was designed and synthesized. In the case of μ- 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.

Original languageEnglish
Pages (from-to)20775-20785
Number of pages11
JournalJournal of Biological Chemistry
Volume279
Issue number20
DOIs
Publication statusPublished - May 14 2004

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Calpain
Substrates
Amino Acids
Mutagenesis
Site-Directed Mutagenesis
Autolysis
Enzyme Inhibitors
Fluorescent Dyes
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Tompa, P., Buzder-Lantos, P., Tantos, A., Farkas, A., Szilágyi, A., Bánóczi, Z., ... Friedrich, P. (2004). On the sequential determinants of calpain cleavage. Journal of Biological Chemistry, 279(20), 20775-20785. https://doi.org/10.1074/jbc.M313873200

On the sequential determinants of calpain cleavage. / Tompa, Peter; Buzder-Lantos, Peter; Tantos, Agnes; Farkas, Attila; Szilágyi, András; Bánóczi, Z.; Hudecz, F.; Friedrich, P.

In: Journal of Biological Chemistry, Vol. 279, No. 20, 14.05.2004, p. 20775-20785.

Research output: Contribution to journalArticle

Tompa, P, Buzder-Lantos, P, Tantos, A, Farkas, A, Szilágyi, A, Bánóczi, Z, Hudecz, F & Friedrich, P 2004, 'On the sequential determinants of calpain cleavage', Journal of Biological Chemistry, vol. 279, no. 20, pp. 20775-20785. https://doi.org/10.1074/jbc.M313873200
Tompa P, Buzder-Lantos P, Tantos A, Farkas A, Szilágyi A, Bánóczi Z et al. On the sequential determinants of calpain cleavage. Journal of Biological Chemistry. 2004 May 14;279(20):20775-20785. https://doi.org/10.1074/jbc.M313873200
Tompa, Peter ; Buzder-Lantos, Peter ; Tantos, Agnes ; Farkas, Attila ; Szilágyi, András ; Bánóczi, Z. ; Hudecz, F. ; Friedrich, P. / On the sequential determinants of calpain cleavage. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 20. pp. 20775-20785.
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