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Abstract
The cysteine protease CP14 has been identified as a central component of a molecular module regulating programmed cell death in plant embryos. CP14 belongs to a distinct subfamily of papain-like cysteine proteinases of which no representative has been characterized thoroughly to date. However, it has been proposed that CP14 is a cathepsin H-like protease. We have now produced recombinant Nicotiana benthamiana CP14 (NbCP14) lacking the C-terminal granulin domain. As typical for papain-like cysteine proteinases, NbCP14 undergoes rapid autocatalytic activation when incubated at low pH. The mature protease is capable of hydrolysing several synthetic endopeptidase substrates, but cathepsin H-like aminopeptidase activity could not be detected. NbCP14 displays a strong preference for aliphatic over aromatic amino acids in the specificity-determining P2 position. This subsite selectivity was also observed upon digestion of proteome-derived peptide libraries. Notably, the specificity profile of NbCP14 differs from that of aleurain-like protease, the N. benthamiana orthologue of cathepsin H. We conclude that CP14 is a papain-like cysteine proteinase with unusual enzymatic properties which may prove of central importance for the execution of programmed cell death during plant development.
Highlights
The tobacco-related plant species Nicotiana benthamiana is a popular model organism for the study of plant-pathogen interactions [1]
Substantial evidence has been obtained for a molecular arms race between plant-derived papain-like cysteine proteinases (PLCPs) and antagonistic effectors released by Abbreviations: ALP, aleurain-like protease; Bz, benzoyl; CHN2, diazomethyl; CDR, complementarity-determining region; DTT, dithiothreitol; mAb, monoclonal antibody; MCA, 4-methylcoumaryl-7-amide; Nb, Nicotiana benthamiana; PICS, Proteomic Identification of Protease Cleavage Sites; PLCP, papain-like cysteine proteinase; Z, benzyloxycarbonyl
Full-length Nicotiana benthamiana CP14 (NbCP14) (505 amino acids; 56.9 kDa; GenBank: KU212214) shares 93% identity with NtCP14 (GenBank: KF113573), and the catalytic domains differ only at 11 positions (5%). Both enzymes belong to PLCP subfamily 4 [17] and are considerably larger than other PLCPs due to the additional presence of a C-terminal granulin domain (108 amino acids), which is connected to the other parts of the protease by a 30-residue proline-rich linker region
Summary
The tobacco-related plant species Nicotiana benthamiana is a popular model organism for the study of plant-pathogen interactions [1]. Only two N. benthamiana PLCPs have been studied thoroughly on the protein level so far: aleurain-like protease (ALP), assigned to subfamily 8, and cathepsin B, a member of subfamily 9 [19e21]. Another N. benthamiana PLCP has recently received substantial attention. To study the catalytic features of N. benthamiana NbC14 (hereafter referred to as NbCP14) in depth, we have produced recombinant forms of this protease in different heterologous expression systems This enabled us to conduct a detailed characterization of its enzymatic properties using synthetic substrates as well as proteome-derived peptide libraries. Our results demonstrate that CP14 lacks cathepsin H-like aminopeptidase activity and exhibits a specificity profile distinct of ALP, the plant orthologue of cathepsin H
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