title: Nicotiana benthamiana cathepsin B displays distinct enzymatic features which differ from its human relative and aleurain-like protease.
abstract: The tobacco-related plant species Nicotiana benthamiana has recently emerged as a versatile expression platform for the rapid generation of recombinant biopharmaceuticals, but product yield and quality frequently suffer from unintended proteolysis. Previous studies have highlighted that recombinant protein fragmentation in plants involves papain-like cysteine proteinases (PLCPs). For this reason, we have now characterized two major N. benthamiana PLCPs in detail: aleurain-like protease (NbALP) and cathepsin B (NbCathB). As typical for PLCPs, the precursor of NbCathB readily undergoes autocatalytic activation when incubated at low pH. On the contrary, maturation of NbALP requires the presence of a cathepsin L-like PLCP as processing enzyme. While the catalytic features of NbALP closely resemble those of its mammalian homologue cathepsin H, NbCathB displays remarkable differences to human cathepsin B. In particular, NbCathB appears to be a far less efficient peptidyldipeptidase (removing C-terminal dipeptides) than its human counterpart, suggesting that it functions primarily as an endopeptidase. Importantly, NbCathB was far more efficient than NbALP in processing the human anti-HIV-1 antibody 2F5 into fragments observed during its production in N. benthamiana. This suggests that targeted down-regulation of NbCathB could improve the performance of this plant-based expression platform.
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: Niemer
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Mehofer
authors.affiliation: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Maria
authors.initials: M
authors.lastname: Verdianz
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: Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Philipp
authors.initials: P
authors.lastname: Schähs
authors.affiliation: Department of Food Science and Technology, University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kracher
authors.affiliation: Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia.
authors.forename: Brigita
authors.initials: B
authors.lastname: Lenarcic
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: 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: D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D015153:Blotting, Western; D002401:Cathepsin B / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010447:Peptide Hydrolases / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D011994:Recombinant Proteins / Q000378:metabolism; D061987:Sf9 Cells; D018411:Spodoptera; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology*
chemical_list: D004792:Enzyme Precursors; D010940:Plant Proteins; D011994:Recombinant Proteins; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; C046726:aleurain; D002401:Cathepsin B
keywords: Antibody; Cathepsin; Cysteine protease; Expression platform; Plant; Proteolysis