Abstract
Calpains are intracellular Ca2+-regulated cysteine proteases that are essential for various cellular functions. Mammalian conventional calpains (calpain-1 and calpain-2) modulate the structure and function of their substrates by limited proteolysis. Thus, it is critically important to determine the site(s) in proteins at which calpains cleave. However, the calpains' substrate specificity remains unclear, because the amino acid (aa) sequences around their cleavage sites are very diverse. To clarify calpains' substrate specificities, 84 20-mer oligopeptides, corresponding to P10-P10′ of reported cleavage site sequences, were proteolyzed by calpains, and the catalytic efficiencies (kcat/Km) were globally determined by LC/MS. This analysis revealed 483 cleavage site sequences, including 360 novel ones. The kcat/Kms for 119 sites ranged from 12.5–1,710 M−1s−1. Although most sites were cleaved by both calpain-1 and −2 with a similar kcat/Km, sequence comparisons revealed distinct aa preferences at P9-P7/P2/P5′. The aa compositions of the novel sites were not statistically different from those of previously reported sites as a whole, suggesting calpains have a strict implicit rule for sequence specificity, and that the limited proteolysis of intact substrates is because of substrates' higher-order structures. Cleavage position frequencies indicated that longer sequences N-terminal to the cleavage site (P-sites) were preferred for proteolysis over C-terminal (P′-sites). Quantitative structure-activity relationship (QSAR) analyses using partial least-squares regression and >1,300 aa descriptors achieved kcat/Km prediction with r = 0.834, and binary-QSAR modeling attained an 87.5% positive prediction value for 132 reported calpain cleavage sites independent of our model construction. These results outperformed previous calpain cleavage predictors, and revealed the importance of the P2, P3′, and P4′ sites, and P1-P2 cooperativity. Furthermore, using our binary-QSAR model, novel cleavage sites in myoglobin were identified, verifying our predictor. This study increases our understanding of calpain substrate specificities, and opens calpains to “next-generation,” i.e. activity-related quantitative and cooperativity-dependent analyses.
Highlights
From the ‡Calpain Project, Department of Advanced Science for Biomolecules, and §The Advanced Technical Support Department, The Basic Technology Research Center, Tokyo Metropolitan Institute of Medical Science (IGAKUKEN), 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan; ¶Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8561, Japan; ʈGraduate School of Information Science and Technology, Hokkaido University, Kita 14, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-0814, Japan; **Bioinformatics Center, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
We found that 98 of the 131 Rp sites existing in the P87mix were proteolyzed by calpains (74 Rp sites were in the middle of the peptide [i.e. after position 10], and 70 of these were proteolyzed), even using oligopeptides, indicating that the calpain substrate specificity was consistent and validating our experimental system
MS and peptide sequencing analyses revealed that two of the three identified Nv sites [C-terminal to Phe80 and Leu84] were detected. This experiment showed that some of the Nv sites, if not all, are cleaved by calpains in full-length proteins, and they have just not been reported yet. These results strongly suggested that the calpains did not randomly proteolyze the oligopeptide mixture, but that all of the detected proteolytic sites strictly complied with an as-yetunknown rule for calpain substrate specificity
Summary
Peptides and Calpains—From 116 reports, 147 calpain substrates, and their 420 cleavage-site sequences (after excluding two overlapping sequences from a total of 422) were collected (supplemental Table S1). Peptides corresponding to 39 (C-terminal) and 15 (N-terminal) fragments obtained by cleavages at Nv sites were synthesized (supplemental Table S3, ID0XX-Nv series) These peptides (158 total peptides, named “P158mix”) were used to quantify the generated calpain-cleaved peptides in the following kinetics experiments. Peptides were identified using ProteinPilotTM Ver.4.5 with the following Paragon parameters: Sample Type: iTRAQ 8plex (Peptide Labeled); Cys Alkylation: MMTS; Digestion: None; Instrument: QSTAR Elite ESI or 4800; Special Factors: “N-Ac and C-DKP” or “N-Ac and C-DKP, cleavable” (see below); Species: None; Specify Processing: check in Quantitate, Bias Correction, Background Correction, Biological modifications; Search Effort: Thorough ID; Results Quality: Detected Protein Threshold Ͼ 0.05 and Run False Discovery Rate Analysis (Threshold Ͼ 0.05 is recommended by the manufacturer, and the FDR was calculated automatically by ProteinPilotTM); Database: Hs4K DB (normal condition) or Hs50K DB (stringent condition). For the standard aa compositions, the following values taken from Swiss-Prot DB release 2012_9 were used: Ala, 8.67; Cys, 1.26; Asp, 5.32; Glu, 6.17; Phe, 4.01; Gly, 7.10; His, 2.21; Ile, 5.96; Lys, 5.25; Leu, 9.92; Met, 2.46; Asn, 4.09; Pro, 4.71; Gln, 3.95; Arg, 5.46; Ser, 6.66; Thr, 5.57; Val, 6.77; Trp, 1.30; Tyr, 3.03 (%)
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