On the Sequential Determinants of Calpain Cleavage
2004; Elsevier BV; Volume: 279; Issue: 20 Linguagem: Inglês
10.1074/jbc.m313873200
ISSN1083-351X
AutoresPéter Tompa, Péter Buzder-Lantos, Ágnes Tantos, Attila E. Farkas, András Szilágyi, Zoltán Bánóczi, Ferenc Hudecz, Péter Friedrich,
Tópico(s)Signaling Pathways in Disease
ResumoThe 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′-P4′; 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, DABCYLTPLKSPPPSPR-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. 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′-P4′; 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, DABCYLTPLKSPPPSPR-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. It is generally held that calpains, intracellular cysteine proteases, play crucial roles in basic physiological (1Sorimachi H. Ishiura S. Suzuki K. Biochem. J. 1997; 328: 721-732Crossref PubMed Scopus (621) Google Scholar, 2Ono Y. Sorimachi H. Suzuki K. Biochem. Biophys. Res. Commun. 1998; 245: 289-294Crossref PubMed Scopus (107) Google Scholar, 3Carafoli E. Molinari M. Biochem. Biophys. Res. Commun. 1998; 247: 193-203Crossref PubMed Scopus (341) Google Scholar) and pathological (4Huang Y. Wang K.K. Trends Mol. Med. 2001; 7: 355-362Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar) processes. This view is partially based on the fact that calpains cleave their substrates in a limited manner, modifying, rather than terminating, their action. So far, more than 100 proteins have been identified as calpain substrates; among them, cytoskeletal/structural proteins, membrane receptors, enzymes, and transcription factors abound (5Wang K.K. Villalobo A. Roufogalis B.D. Biochem. J. 1989; 262: 693-706Crossref PubMed Scopus (247) Google Scholar, 6Wang K.K.W. Yuen P.-w. Wang K.K.W. Yuen P.-w. Calpain: Pharmacology and Toxicology of Calcium-dependent Protease. Taylor and Francis, Philadelphia1999: 77-101Google Scholar). Despite this wealth of data, the sequential/structural determinants of calpain cleavage are little understood. Early studies on small fluorogenic peptides (7Sasaki T. Kikuchi T. Yumoto N. Yoshimura N. Murachi T. J. Biol. Chem. 1984; 259: 12489-12494Abstract Full Text PDF PubMed Google Scholar) and peptide hormones (7Sasaki T. Kikuchi T. Yumoto N. Yoshimura N. Murachi T. J. Biol. Chem. 1984; 259: 12489-12494Abstract Full Text PDF PubMed Google Scholar, 8Hirao T. Takahashi K. J. Biochem. (Tokyo). 1984; 96: 775-784Crossref PubMed Scopus (30) Google Scholar) have led to the so-called P2-P1 rule, which states that the preferred residues for the ubiquitous forms, μ- and m-calpain, are Leu and Val at position P2, whereas in P1 Arg and Lys (and to a lesser extent Tyr) occur most often (9Takahashi K. Mellgren R.L. Murachi T. Calpain Substrate Specificity. CRC Press, Boca Raton1990Google Scholar). Additional, weaker, preferences have also been noted for positions P3 (Phe, Trp, Leu, Val) and P1′ (Arg, Lys, Leu). An elegant study encompassing all possible point mutations at the P2 position of the principal cleavage site of fodrin has shown good correlation with the P2-P1 rule (10Stabach P.R. Cianci C.D. Glantz S.B. Zhang Z. Morrow J.S. Biochemistry. 1997; 36: 57-65Crossref PubMed Scopus (69) Google Scholar); other studies on protein substrates, however, displayed frequent deviations (11Sakai K. Akanuma H. Imahori K. Kawashima S. J. Biochem. (Tokyo). 1987; 101: 911-918Crossref PubMed Scopus (9) Google Scholar, 12Banik N.L. Chou C.H. Deibler G.E. Krutzch H.C. Hogan E.L. J. Neurosci. Res. 1994; 37: 489-496Crossref PubMed Scopus (41) Google Scholar). These observations suggested that calpain cleavage, to a large extent, depends on higher order structural clues. This aspect of substrate recognition was convincingly demonstrated in histone hydrolysis, where cleavage of sites susceptible in the intact protein was restrained in its fragments (11Sakai K. Akanuma H. Imahori K. Kawashima S. J. Biochem. (Tokyo). 1987; 101: 911-918Crossref PubMed Scopus (9) Google Scholar). It was concluded that calpain probably recognizes global structural elements present in the intact protein but absent from its smaller fragments. This recognition has fueled efforts to uncover these putative higher order structural elements. In the above report (11Sakai K. Akanuma H. Imahori K. Kawashima S. J. Biochem. (Tokyo). 1987; 101: 911-918Crossref PubMed Scopus (9) Google Scholar) it was suggested that cleavage, as in histone, may never occur in the middle of either an hydrophobic or hydrophilic cluster but always at the border separating them. In another, comprehensive treatise including most protein substrates known at the time (5Wang K.K. Villalobo A. Roufogalis B.D. Biochem. J. 1989; 262: 693-706Crossref PubMed Scopus (247) Google Scholar) it was found that calpain substrates often have a calmodulin-binding motif and targeting of the enzyme may occur by an adjacent PEST 1The abbreviations used are: PEST, Pro, Glu(Asp), and Ser/Thr region; DABCYL, 4-(4-dimethylaminophenylazo)benzoic acid; EDANS, 5-[(2-aminoethyl) amino]naphthalene-1-sulfonic acid; FRET, Förstertype resonance energy transfer; LY-AMC, N-succinyl-Leu-Tyr-7-amido-4-methylcoumarine; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; RP-HPLC, reverse phase high performance liquid chromatography; BOC, tert-butyloxycarbonyl; Fmoc, 9-fluorenylmethoxycarbonyl; DMF, N,N-dimethylformamide. sequence. This idea was raised in the original proposal of the PEST hypothesis, i.e. that the occurrence of region(s) rich in amino acids Pro, Glu(Asp), and Ser/Thr flanked by Arg/Lys residues correlates with the short lifetime of proteins (13Rogers S. Wells R. Rechsteiner M. Science. 1986; 234: 364-368Crossref PubMed Scopus (1964) Google Scholar, 14Rechsteiner M. Rogers S.W. Trends Biochem. Sci. 1996; 21: 267-271Abstract Full Text PDF PubMed Scopus (1413) Google Scholar). Such a negatively charged region was thought to bind Ca2+ and provide both peptide bonds and the necessary co-factor for calpain action. Subsequent experimental testing of these suggestions brought controversial results, though. Purification of μ-calpain by an immobilized peptide containing the calmodulin-binding motif of plasma membrane ATPase supported the involvement of such region(s) (15Molinari M. Maki M. Carafoli E. J. Biol. Chem. 1995; 270: 14576-14581Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Attempts with the PEST motif, however, yielded equivocal results. In some instances mutation of the PEST sequence abolished calpain cleavage (16Noguchi M. Sarin A. Aman M.J. Nakajima H. Shores E.W. Henkart P.A. Leonard W.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11534-11539Crossref PubMed Scopus (71) Google Scholar, 17Shumway S.D. Maki M. Miyamoto S. J. Biol. Chem. 1999; 274: 30874-30881Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) but was without effect in other cases (18Molinari M. Anagli J. Carafoli E. J. Biol. Chem. 1995; 270: 2032-2035Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19Carillo S. Pariat M. Steff A. Jariel-Encontre I. Poulat F. Berta P. Piechaczyk M. Biochem. J. 1996; 313: 245-251Crossref PubMed Scopus (52) Google Scholar). Thus, whether a PEST region provides a degradation signal for calpain, is still an open issue. In general, higher order clues still lay largely hidden in the structure of calpain substrates, and recent reviews on the subject (2Ono Y. Sorimachi H. Suzuki K. Biochem. Biophys. Res. Commun. 1998; 245: 289-294Crossref PubMed Scopus (107) Google Scholar, 6Wang K.K.W. Yuen P.-w. Wang K.K.W. Yuen P.-w. Calpain: Pharmacology and Toxicology of Calcium-dependent Protease. Taylor and Francis, Philadelphia1999: 77-101Google Scholar, 20Wang K.K. Trends Neurosci. 2000; 23: 20-26Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar) resort to the P2-P1 rule unveiled a long time ago. In our view, the major drawback of this point of reference is that it is mostly based on studies with peptides, known to be poor calpain substrates (9Takahashi K. Mellgren R.L. Murachi T. Calpain Substrate Specificity. CRC Press, Boca Raton1990Google Scholar). Further, the use of a small number of peptides limits the range of amino acid combinations tested, and thus biases the amino acid preferences observed. An incomparably larger and more complete set of potential substrate sites is represented by the proteome, of which the cleavable ones make their way into the literature as calpain substrates. To advance our understanding of what constitutes a calpain site, we carried out a systematic analysis of these data and constructed a preference matrix. The sequential preferences manifest in this matrix point to some general aspects of the primary structure in substrate recognition by μ- and m-calpain. Furthermore, the versatile applicability of this matrix in engineering substrates of these ubiquitous calpains is also described in this report. Data Collection and Analysis—49 calpain substrates with a total of 106 sequentially identified cleavage sites have been selected from the literature. For homologous substrates cleaved at identical sites, only one species (preferably human) was considered. The protein sequences were downloaded from the Swiss-Prot and TrEMBL databases via the ExPASy molecular biology server (www.expasy.ch). The sequences were truncated to 50 amino acids in both the N- and C-terminal direction from the scissile bond and used for further studies. Amino acid frequency matrices and profile scores were calculated by a program written in the Python programming language. The amino acid composition of the complete Swiss-Prot and TrEMBL data base used for normalizing matrices was taken from Swiss-Prot (us.expasy.org/sprot/). Sequential attributes were calculated or predicted by the appropriate servers: PEST-score with the PESTfind algorithm (www.at.embnet.org/embnet/tools/bio/PESTfind), secondary structure with the nnPredict site (www.cmpharm.ucsf.edu/∼nomi/nnpredict.html), and hydrophobicity with ProtScale (us.expasy.org/cgi-bin/protscale.pl). These servers are available via ExPASy. For strong and weak PEST regions detected by the PESTfind algorithm, we arbitrarily assigned a score of 3 and 1. For scoring the border of a weak PEST region, its flanking hexapeptide was assigned weights of 1, 2, 4, 4, 2, and 1. For a strong PEST region, weights of 1, 3, 9, 9, 3, and 1 were used; in both cases all other residues were set to 0. Synthesis of FRET Substrate—The substrate, designed on the basis of the observed amino acid preferences of calpain cleavage, is an 11-mer peptide with the sequence of Thr-Pro-Leu-Lys-Ser-Pro-Pro-Pro-Ser-Pro-Arg, with a fluorescent donor, EDANS (Sigma) attached to its C terminus and a quenching acceptor, DABCYL attached to the N terminus. Since the measurement of calpain activity is based on the increase of donor fluorescence due to cessation of FRET between the EDANS and DABCYL groups upon cleavage (21Matayoshi E.D. Wang G.T. Krafft G.A. Erickson J. Science. 1990; 247: 954-958Crossref PubMed Scopus (562) Google Scholar), this substrate is termed as the FRET substrate throughout the article. The peptide was synthesized on 0.2 g of Boc-Arg(Mts)-PAM-resin (0.31 mmol/g) (from NovaBiochem, Laufelfingen, Switzerland) by Boc (tert-butyloxycarbonyl) chemistry. Side chains of threonine and serines were protected with benzyl groups, whereas the ϵ-amino group of lysine was blocked by an Fmoc (9-fluorenylmethoxycarbonyl) group. (All Boc amino acid derivatives were obtained from Reanal, Budapest, Hungary.) Boc protection was removed with 33% trifluoroacetic acid in DCM (2 + 20 min) followed by washing with DCM (5 × 0.5 min), neutralization with 10% DIEA in DCM (3 × 1 min), and DCM wash (4 × 0.5 min). The amino acid derivatives and coupling reagents (N,N,N′-dicyclohexyl carbodiimide and 1-hydroxybenzotriazole, at 1:1 molar ratio) dissolved in DCM-DMF (N,N-dimethylformamide) 4:1 (v/v) were used in 3 molar excess for the resin capacity. The coupling reaction was continued for 60 min at room temperature, and the resin was washed with DMF (2 × 0.5 min) and DCM (3 × 0.5 min). The efficiency of coupling was checked by ninhydrin or isatin tests. The N-terminal Boc group was removed from the protected peptide prior to cleavage by hydrogen-fluoride. The peptide was removed from the resin with 10 ml of hydrogen fluoride containing 0.5 g of p-cresol. The crude product was purified by RP-HPLC. DABCYL was coupled to the N-terminal amino group of the purified H-TPLK(Fmoc) SPPPSPR-OH peptide in solution. Peptide was dissolved in DMF and reacted with DABCYL succinimide ester (Fluka, Buchs, Switzerland) in the presence of DIEA (DABCYL succinimide/DIEA/peptide = 2:1:1 mol/mol/mol). The crude product was purified by RP-HPLC. The Nϵ-Fmoc group of the purified DABCYL-TPLK(Fmoc)SPPPSPR-OH peptide was removed with 20% piperidine/DMF. The peptide was purified by RP-HPLC. EDANS was attached to the C terminus of the DABCYL-peptide in DMF, using the following molar ratio: peptide/EDANS/EDC×HCl/1-hydroxybenzotriazole/DIEA = 1:2:4.5:6.8:9 (mol/mol). The target peptide was purified by RP-HPLC. The peptides were characterized by analytical HPLC (see below), amino acid analysis using a Beckman (Fullerton, CA) model 6300 amino acid analyzer and by mass spectra recorded on a PerkinElmer Life Sciences API 2000 triple quadrupole mass spectrometer (Sciex, Toronto, Canada). Analytical RP-HPLC was performed on a Knauer (Bad Homburg, Germany) HPLC system using Phenomenex Synergi MAX-RP C12 column (250 × 4.6 inner diameter) with 5-mm silica (80 Å pore size) as a stationary phase. Linear gradient elution (0 min, 0% B; 5 min, 0% B; 50 min, 90% B) with eluent A (0.1% trifluoroacetic acid in water) and eluent B (0.1% trifluoroacetic acid in acetonitrile/water (80:20, v/v)) was used at a flow rate of 1 ml/min at ambient temperature. Sample was dissolved in eluent B, and peaks were detected at a λ of 220 nm. The crude product was purified on a semipreparative Phenomenex Jupiter C18 column (250 × 10-mm inner diameter) with silica (300 Å pore size). The peptides were checked by amino acid analysis using a Model 6300 amino acid analyzer, and mass spectra were recorded on an API 2000 triple quadrupole mass spectrometer. Calpain B Mutagenesis and Autolysis Experiments—Calpain B autolysis sites (22Farkas A. Tompa P. Schad E. Sinka R. Jekely G. Friedrich P. Biochem. J. 2004; 378: 299-305Crossref PubMed Google Scholar) were mutated with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's recommendations using the following primers: 5′-CGAGTCATGCCGGTGTTCCGTCGTATGCGGGAC-3′ and 5′-GTCCCGCATACGACGGAACACCGGCATGACTCG-3′ for the first major site; 5′-GAAGGTGCCCGAGGCTGTTAACATGTTTTGG-3′ and 5′-CAAAACATGTTAACAGCCTCGGGCACCTTC-3′ for the second major site (22Farkas A. Tompa P. Schad E. Sinka R. Jekely G. Friedrich P. Biochem. J. 2004; 378: 299-305Crossref PubMed Google Scholar). Calpain B autolysis reaction was started in calpain buffer (10 mm HEPES, pH 7.5, 150 mm NaCl, and 1 mm EDTA) with 10 mm free Ca2+ and run for 10 min. The reaction was stopped by the addition of SDS sample buffer and 5 min of boiling. Samples were run on an SDS-polyacrylamide gel. Enzyme Activity Measurements—Activity measurements were carried out with m-calpain, calpain A, calpain B, papain, trypsin, chymotrypsin, and cathepsin-B. Enzyme activity was measured with a Jasco FP 777 spectrofluorometer at excitation/emission wavelengths of 380/460 nm for LY-AMC (Sigma) and 320/480 nm for the FRET substrate, in a 3 × 3 mm quartz cuvette. The reaction mixture in 50 μl of calpain buffer (10 mm HEPES, 150 mm NaCl, 1 mm EDTA, 5 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluoride, 10 mm β-mercaptoethanol, pH 7.5) contained various substrate concentrations and 3 mm free Ca2+ in the case of m-calpain and 19 mm free Ca2+ in the case of calpain A and calpain B. The reaction was started by rapidly mixing the enzyme in the reaction mixture. The enzyme concentrations typically used were as follows: 0.2 μm m-calpain, 4.8 μm calpain A, 1.8 μm calpain B, 1.0 μm papain, 1.0 μm trypsin, 40.0 μm chymotrypsin, and 10.0 μm cathepsin B. Data were analyzed by the MicroCal Origin data analysis software to determine the initial slope of fluorescence change. Calpain Activity Measurements in Vivo—In vivo calpain activity was measured in cultured Drosophila S2 cells. The cell cultures were maintained in Drosophila SFM (Invitrogen) medium supplemented with 20 mm l-glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin at 23 °C. Four hours prior to starting the experiments, 200 μl of S2 cells (3 × 106 cell/ml) were lipofected with 200 μl of FRET substrate (giving a final substrate concentration of 280 μm in the medium) either without or with 15 μm calpastatin, using Cellfectin (Invitrogen) reagent. The protocol provided by the manufacturer was followed. Control cells were treated with the same amount of Cellfectin, but without the addition of the FRET substrate. Before measurement, lipofected cells were collected by centrifugation at 1000 rpm for 4 min at room temperature and were resuspended in 400 μl of phosphate-buffered saline. This washing procedure was repeated twice to remove all the remaining substrate from the medium. 50 μl of cell suspension was transferred to a 3 × 3 mm quartz cuvette for recording fluorescence. Measurements with lipofected and control cells were started by the addition of 10 μm ionomycin either with or without 500 nm E-64 or calpain inhibitor II (both are cell permeable calpain inhibitors). The fluorescence measurements and data analysis were done similarly to the in vitro experiments. COS-7 Cell Extracts—COS-7 cells were grown in Dulbecco's modified Eagle's medium (Sigma) at 37 °C and 5% CO2 content. Confluent cells were washed three times with phosphate-buffered saline and were scraped in 1 ml of ice-cold phosphate-buffered saline. Cells were collected by centrifugation at 1500 × g for 2 min at 4 °C. Collected cells were resuspended in a buffer containing 100 mm Tris-HCl, 5 mm EDTA, 1 mm dithioerythritol, 5 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluoride, and 10 mm β-mercaptoethanol and were sonicated four times for 10 s with 1-min breaks. After sonication, the lysate was centrifuged at 15,000 × g for 20 min at 4 °C to remove the cell debris, and the supernatant was used for fluorometric calpain activity measurements. Proteins—The 80-kDa large subunit and the 21-kDa truncated small subunit of rat m-calpain was expressed in Escherichia coli and purified according to the method described in Ref. 23Elce J.S. Hegadorn C. Gauthier S. Vince J.W. Davies P.L. Protein Eng. 1995; 8: 843-848Crossref PubMed Scopus (65) Google Scholar. Drosophila calpain A and calpain B were expressed in E. coli using the method described in Ref. 24Jekely G. Friedrich P. J. Biol. Chem. 1999; 274: 23893-23900Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar. Human calpastatin domain 1 was prepared as given in Ref. 25Yang H.Q. Ma H. Takano E. Hatanaka M. Maki M. J. Biol. Chem. 1994; 269: 18977-18984Abstract Full Text PDF PubMed Google Scholar. Other enzymes for activity measurements, such as papain (P-3375), trypsin (T-8003), α-chymotrypsin (C-4129), and cathepsin B (C-6286), were purchased from Sigma. These lyophilized enzymes were dissolved in calpain buffer just prior to the experiment and were kept on ice until use. Other Methods and Materials—SDS-polyacrylamide gel electrophoresis was carried out according to Ref. 26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar. Calpain inhibitors were purchased from Calbiochem. For N-terminal sequence analysis, samples were run on SDS-PAGE gels and blotted onto polyvinylidene difluoride membranes (Sigma). N-terminal sequence analysis was performed using a modified Edman degradation sequencer program. The Preference Matrix—The 106 sites of 49 substrates are listed in Table I. The number of proteins known to be calpain substrates is actually significantly higher than this. For most proteins, however, the exact site of cleavage has not been determined. Cleavage by μ- and m-calpain has not been distinguished, because the two ubiquitous isoforms have nearly identical substrate preferences (6Wang K.K.W. Yuen P.-w. Wang K.K.W. Yuen P.-w. Calpain: Pharmacology and Toxicology of Calcium-dependent Protease. Taylor and Francis, Philadelphia1999: 77-101Google Scholar, 9Takahashi K. Mellgren R.L. Murachi T. Calpain Substrate Specificity. 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J. 1987; 248: 579-588Crossref PubMed Scopus (29) Google ScholarAlpain 80KChickenArg-Leu-Arg17-Ala-Glu-Gly51Crawford C. Willis A.C. Gagnon J. Biochem. J. 1987; 248: 579-588Crossref PubMed Scopus (29) Google ScholarCaMK IVMouseVal-Cys-Gly201-Thr-Pro-Gly, Thr-Glu-Asn23-Leu-Val-Pro52McGinnis K.M. Whitton M.M. Gnegy M.E. Wang K.K. J. Biol. Chem. 1998; 273: 19993-20000Abstract Full Text Full Text PDF PubMed Scopus (92) Google ScholarCaM-PDE1A2BovineVal-Val-Gln126-Ala-Gly-Ile53Kakkar R. Raju R.V. Sharma R.K. Arch. Biochem. Biophys. 1998; 358: 320-328Crossref PubMed Scopus (21) Google ScholarCaspase-9HumanGln-Leu-Asp330-Ala-Ile-Ser, Pro-Glu-Ile115-Arg-Lys-Pro54Chua B.T. Guo K. Li P. J. Biol. Chem. 2000; 275: 5131-5135Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholarc-FosRatSer-Gln-Thr90-Arg-Ala-Pro55Pariat M. Salvat C. Bebien M. Brockly F. Altieri E. Carillo S. Jariel-Encontre I. Piechaczyk M. Biochem. 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