Journal
BIOLOGICAL CHEMISTRY
Volume 399, Issue 10, Pages 1223-1235Publisher
WALTER DE GRUYTER GMBH
DOI: 10.1515/hsz-2018-0186
Keywords
cysteine proteinase; degradomics; exopeptidase; plant; substrate profiling
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Funding
- Austrian Science Fund (FWF) [W1224-B09]
- Deutsche Forschungsgemeinschaft (DFG) [SCHI 871/2, SCHI 871/5, SCHI 871/6, GR 1748/6, INST 39/900-1, SFB850]
- European Research Council [ERC-2011-StG 282111-ProteaSys]
- Excellence Initiative of the German Federal and State Governments (BIOSS) [EXC 294]
- BOKU Equipment GmbH
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The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly(336)-> Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxy-dipeptidase determinant His(207) also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.
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