Profiling of calpain activity with a series of FRET-based substrates.
Calpains are intracellular proteases that selectively cleave proteins in response to calcium signals. Although calpains cut many different sequences, residue preferences within peptide substrates were recently determined and incorporated into a superior FRET (fluorescence resonance energy transfer)-based substrate (PLFAER). Here we show PLFAER is cleaved by calpain at the intended F-A scissile bond. Sequential replacement of individual residues by alanine reduced activity except with PLFAAR, which is cleaved 2.3 times faster than PLFAER. The rates of hydrolysis of the alanine-substituted substrates were used to compare substrate preferences of calpain, papain and cathepsins B and L. The preferences of the two major isoforms, calpains 1 and 2, were virtually indistinguishable and were very similar to those of the calpain 1 protease core and papain. However, the activity profiles with the FRET substrate series were significantly different for the cathepsins, particularly cathepsin B.
Kelly
Jacqueline C
JC
Department of Biochemistry, Queen's University, Kingston, ON, Canada K7L 3N6.
Cuerrier
Dominic
D
Graham
Laurie A
LA
Campbell
Robert L
RL
Davies
Peter L
PL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002154:Calpain / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006868:Hydrolysis; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009282:Naphthalenesulfonates; D009842:Oligopeptides / Q000737:chemistry; D010206:Papain / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D005456:Fluorescent Dyes; D007527:Isoenzymes; D009282:Naphthalenesulfonates; D009842:Oligopeptides; D011994:Recombinant Proteins; C063126:5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid; C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004124:p-Dimethylaminoazobenzene; D002403:Cathepsins; D002154:Calpain; D010206:Papain