title: The papain-like cysteine proteinases NbCysP6 and NbCysP7 are highly processive enzymes with substrate specificities complementary to Nicotiana benthamiana cathepsin B.
abstract: The tobacco-related plant Nicotiana benthamiana is gaining interest as a versatile host for the production of monoclonal antibodies and other protein therapeutics. However, the susceptibility of plant-derived recombinant proteins to endogenous proteolytic enzymes limits their use as biopharmaceuticals. We have now identified two previously uncharacterized N. benthamiana proteases with high antibody-degrading activity, the papain-like cysteine proteinases NbCysP6 and NbCysP7. Both enzymes are capable of hydrolysing a wide range of synthetic substrates, although only NbCysP6 tolerates basic amino acids in its specificity-determining S2 subsite. The overlapping substrate specificities of NbCysP6 and NbCysP7 are also documented by the closely related properties of their other subsites as deduced from the action of the enzymes on proteome-derived peptide libraries. Notable differences were observed to the substrate preferences of N. benthamiana cathepsin B, another antibody-degrading papain-like cysteine proteinase. The complementary activities of NbCysP6, NbCysP7 and N. benthamiana cathepsin B indicate synergistic roles of these proteases in the turnover of recombinant and endogenous proteins in planta, thus representing a paradigm for the shaping of plant proteomes by the combined action of papain-like cysteine proteinases.
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Melanie
authors.initials: M
authors.lastname: Paireder
authors.affiliation: Institute for Molecular Medicine and Cell Research, University of Freiburg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Tholen
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Porodko
authors.affiliation: Institute for Molecular Medicine and Cell Research, University of Freiburg, Germany.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Biniossek
authors.affiliation: Institute for Molecular Medicine and Cell Research, University of Freiburg, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Mayer
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia.
authors.forename: Marko
authors.initials: M
authors.lastname: Novinec
authors.affiliation: Institute for Molecular Medicine and Cell Research, University of Freiburg, Germany; BIOSS Centre for Biological Signaling Studies, University of Freiburg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: lukas.mach@boku.ac.at.
authors.forename: Lukas
authors.initials: L
authors.lastname: Mach
mesh_terms: D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
chemical_list: D010940:Plant Proteins; D002401:Cathepsin B
keywords: Antibody; Cysteine protease; Degradomics; Expression platform; Proteolysis; Substrate profiling