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.

Papain-like cysteine proteases prepare plant cyclic peptide precursors for cyclization

“…Recombinant NbCysP6 Generates Acyclic kB1 from Synthetic LQLK-kB1. NbCysP6 is a cathepsin L-like papain-like cysteine protease (PLCP) from the RD21 subclass (25,26). Like most members of this subclass (25), the domain architecture of NbCysP6 consists of an Nterminal signal peptide, an autoinhibitory I29 domain, a C1 protease domain, a proline-rich segment, and a cysteine-rich C-terminal granulin domain.…”

Low Protease Content in Medicago truncatula Cell Cultures Facilitates Recombinant Protein Production

“…Moreover, it is noteworthy that the papain with the code Medtr1g018840.2 appears both in cell extract and culture medium, which might indicate that this specific protease is in transit within the cell. Furthermore, it has already been established that PLCPs are able to degrade antibody molecules …”

Steric Accessibility of the Cleavage Sites Dictates the Proteolytic Vulnerability of the Anti‐HIV‐1 Antibodies 2F5, 2G12, and PG9 in Plants

“…Using activity‐based probes, enzymatically active forms of the two PLCPs NbALP and NbCysP6 have been detected in the apoplast of N. benthamiana leaves . We have previously shown that recombinant NbALP and NbCysP6 are capable of cleaving the bNAb 2F5 in its CDR H3 loop . However, the insensitivity of 2F5 processing to the general PLCP inhibitor E‐64 contradicts a major contribution of NbALP and NbCysP6 to CDR H3 cleavage in planta.…”

“…[41] We have previously shown that recombinant NbALP and NbCysP6 are capable of cleaving the bNAb 2F5 in its CDR H3 loop. [44,45] However, the insensitivity of 2F5 processing to the general PLCP inhibitor E-64 [20] contradicts a major contribution of NbALP and NbCysP6 to CDR H3 cleavage in planta.…”