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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.

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 (Gly336→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 carboxydipeptidase determinant His207 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.