Functional characterization of Nicotiana benthamiana papain-like cysteine proteinases

Background.

Several studies have demonstrated that plants are suitable hosts for the production of protein therapeutics. Unfortunately, the production of recombinant proteins in plants is frequently hampered by unwanted proteolysis (Outchkourov et al., 2003). For recombinant monoclonal antibodies, the generation of substantial amounts of Fab- and Fc-like degradation products has been observed in different plant species (Doran, 2006). This is also the case in Nicotiana benthamiana, a tobacco-related plant species widely used for recombinant protein production. We have now obtained evidence that in planta fragmentation of antibodies is largely due to the action of papain-like cysteine proteinases. However, the N. benthamiana representatives of this family of proteolytic enzymes are so far only poorly characterized.

Aims and methods.

This proposal aims at the identification and detailed characterization of N. benthamiana papain-like cysteine proteinases. Special attention will be given to those enzymes which reside within the apoplast (since degradation of recombinant proteins occurs largely in this compartment) and their capacity to degrade monoclonal antibodies. So far, only one N. benthamiana papain-like cysteine proteinase, cathepsin B (NbCathB), has been studied on the protein level to some extent (Gilroy et al., 2007). The other two cloned sequences, named NbCYP1 and NbCYP2 (Hao et al., 2006) have not yet been further characterized. However, a putative orthologue of NbCYP1 has been recently found in the apoplastic fluid of the related species N. tabacum. In addition, evidence for the presence of at least six other papain-like enzymes in the apoplast of Nicotiana plants has been obtained in the latter study (Delannoy et al., 2008).

Hence, the following approaches will be pursued for a better understanding of the properties of these enzymes:

(1) cDNAs for NbCYP1, NbCYP2, NbCathB and the other six enzymes (exploiting their presumed close homology to the respective N. tabacum proteins) will be cloned by RT-PCR and then expressed in insect cells as already done successfully for other papain-like cysteine proteinases (Müntener et al., 2005). The recombinant enzymes will be purified exploiting exogenously added affinity tags and then analysed for their enzymatic properties using various synthetic substrates. The individual susceptibility of each recombinant proteinase to inhibition by endogenous cysteine proteinase inhibitors (cystatins) will be also established. Special emphasis will be placed on the capacity of the recombinant enzymes to degrade antibodies. For this, a monoclonal antibody which is particularly proteolysis-prone when expressed in plants, the broadly neutralizing human anti-HIV-1 antibody 2F5 (Sack et al., 2007), and its far more stable counterpart 2G12 (Strasser et al., 2008) will be produced in mammalian cells. The cleavage products obtained upon digestion of 2F5 and 2G12 with the respective recombinant proteinase will then be characterized by mass spectrometry and N-terminal sequencing.

(2) Recent progress in the development of activity-based probes for papain-like cysteine proteinases has allowed the detection of active species of these enzymes in different plants (van der Hoorn et al., 2004; Gilroy et al., 2007). Biotin-tagged versions of these functional probes can be used for the selective isolation of such proteinases from tissue extracts, as previously demonstrated by us for mammalian cathepsin B and cathepsin L (Montaser et al., 2002). This methodology in combination with proteomic analysis of the captured proteins will be employed to attempt the isolation of any as yet undiscovered papain-like cysteine proteinase(s) from apoplastic exudates of N. benthamiana. The respective cDNAs will be cloned using primers based on the peptide sequences obtained by mass spectrometry and then expressed in insect cells for purification and characterization of the encoded proteins as outlined above.

Delannoy, M., Alves, G., Vertommen, D., Ma, J., Boutry, M., Navarre, C. (2008) Identification of peptidases in Nicotiana tabacum leaf intercellular fluid. Proteomics 8, 2285-2298
Doran, P. M. (2006) Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol. 24, 426-432
Gilroy, E. M., Hein, I., van der Hoorn, R., Boevink, P. C., Venter, E., McLellan, H., Kaffarnik, F., Hrubikova, K., Shaw, J., Holeva, M., López, E. C., Borras-Hidalgo, O., Pritchard, L., Loake, G. J., Lacomme, C., Birch, P. R. (2007) Involvement of cathepsin B in the plant disease resistance hypersensitive response. Plant J. 52, 1-13
Hao, L., Hsiang, T., Goodwin, P. H. (2006) Role of two cysteine proteinases in the susceptible response of Nicotiana benthamiana to Colletotrichum destructivum and the hypersensitive response to Pseudomonas syringae pv. tomato. Plant Sci. 170, 1001-1009
Montaser, M., Lalmanach, G., Mach, L. (2002) CA-074, but not its methyl ester CA-074Me, is a selective inhibitor of cathepsin B within living cells. Biol. Chem. 383, 1305-1308
Müntener, K., Willimann, A., Zwicky, R., Svoboda, B., Mach, L., Baici, A. (2005) Folding competence of N-terminally truncated forms of human procathepsin B. J. Biol. Chem. 280, 11973-11980
Outchkourov, N. S., Rogelj, B., Strukelj, B., Jongsma, M. A. (2003) Expression of sea anemone equistatin in potato. Effects of plant proteases on heterologous protein production. Plant Physiol. 133, 379-390
Sack, M., Paetz, A., Kunert, R., Bomble, M., Hesse, F., Stiegler, G., Fischer, R., Katinger, H., Stoeger, E., Rademacher, T. (2007) Functional analysis of the broadly neutralizing human anti-HIV-1 antibody 2F5 produced in transgenic BY-2 suspension cultures. FASEB J. 21, 1655-1664
Strasser, R., Stadlmann, J., Schaehs, M., Stiegler, G., Quendler, H., Mach, L., Glössl, J., Weterings, K., Pabst, M., Steinkellner, H. (2008) Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnol. J. 6, 392-402
Van der Hoorn, R. A., Leeuwenburgh, M. A., Bogyo, M., Joosten, M. H., Peck, S. C. (2004) Activity profiling of papain-like cysteine proteinases in plants. Plant Physiol. 135, 1170-1178