The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities.

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.

Glycan modulation and sulfoengineering of anti-HIV-1 monoclonal antibody PG9 in plants.

Broadly neutralizing anti-HIV-1 monoclonal antibodies, such as PG9, and its derivative RSH hold great promise in AIDS therapy and prevention. An important feature related to the exceptional efficacy of PG9 and RSH is the presence of sulfated tyrosine residues in their antigen-binding regions. To maximize antibody functionalities, we have now produced glycan-optimized, fucose-free versions of PG9 and RSH in Nicotiana benthamiana. Both antibodies were efficiently sulfated in planta on coexpression of an engineered human tyrosylprotein sulfotransferase, resulting in antigen-binding and virus neutralization activities equivalent to PG9 synthesized by mammalian cells ((CHO)PG9). Based on the controlled production of both sulfated and nonsulfated variants in plants, we could unequivocally prove that tyrosine sulfation is critical for the potency of PG9 and RSH. Moreover, the fucose-free antibodies generated in N. benthamiana are capable of inducing antibody-dependent cellular cytotoxicity, an activity not observed for (CHO)PG9. Thus, tailoring of the antigen-binding site combined with glycan modulation and sulfoengineering yielded plant-produced anti-HIV-1 antibodies with effector functions superior to PG9 made in CHO cells.

The papain-like cysteine proteinases NbCysP6 and NbCysP7 are highly processive enzymes with substrate specificities complementary to Nicotiana benthamiana cathepsin B.

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.

The death enzyme CP14 is a unique papain-like cysteine proteinase with a pronounced S2 subsite selectivity.

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.

Nicotiana benthamiana cathepsin B displays distinct enzymatic features which differ from its human relative and aleurain-like protease.

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.