title: Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array.
abstract: Calpains are intracellular Ca(2+)-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. Thekcat/Kms for 119 sites ranged from 12.5-1,710 M(-1)s(-1) Although most sites were cleaved by both calpain-1 and -2 with a similarkcat/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 achievedkcat/Kmprediction withr= 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.
authors.affiliation: From the ‡Calpain Project, Department of Advanced Science for Biomolecules, and.
authors.forename: Fumiko
authors.initials: F
authors.lastname: Shinkai-Ouchi
authors.affiliation: From the ‡Calpain Project, Department of Advanced Science for Biomolecules, and.
authors.forename: Suguru
authors.initials: S
authors.lastname: Koyama
authors.affiliation: From the ‡Calpain Project, Department of Advanced Science for Biomolecules, and.
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Ono
authors.affiliation: From the ‡Calpain Project, Department of Advanced Science for Biomolecules, and.
authors.forename: Shoji
authors.initials: S
authors.lastname: Hata
authors.affiliation: From the ‡Calpain Project, Department of Advanced Science for Biomolecules, and.
authors.forename: Koichi
authors.initials: K
authors.lastname: Ojima
authors.affiliation: §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;
authors.forename: Mayumi
authors.initials: M
authors.lastname: Shindo
authors.affiliation: ¶Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8561, Japan;
authors.forename: David
authors.initials: D
authors.lastname: duVerle
authors.affiliation: From the ‡Calpain Project, Department of Advanced Science for Biomolecules, and.
authors.forename: Mika
authors.initials: M
authors.lastname: Ueno
authors.affiliation: From the ‡Calpain Project, Department of Advanced Science for Biomolecules, and.
authors.forename: Fujiko
authors.initials: F
authors.lastname: Kitamura
authors.affiliation: From the ‡Calpain Project, Department of Advanced Science for Biomolecules, and.
authors.forename: Naoko
authors.initials: N
authors.lastname: Doi
authors.affiliation: ‖Graduate School of Information Science and Technology, Hokkaido University, Kita 14, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-0814, Japan;
authors.forename: Ichigaku
authors.initials: I
authors.lastname: Takigawa
authors.affiliation: **Bioinformatics Center, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mamitsuka
authors.affiliation: From the ‡Calpain Project, Department of Advanced Science for Biomolecules, and sorimachi-hr@igakuken.or.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sorimachi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002154:Calpain / Q000737:chemistry*; D002384:Catalysis; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism*; D059748:Proteolysis; D021281:Quantitative Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; D002154:Calpain