Identification of peptidases in Nicotiana tabacum leaf intercellular fluid

Abstract

Peptidases in the extracellular space might affect the integrity of recombinant proteins expressed in, and secreted from, plant cells. To identify extracellular peptidases, we recovered the leaf intercellular fluid from Nicotiana tabacum plants by an infiltration-centrifugation method. The activity of various peptidases was detected by an in vitro assay in the presence of specific inhibitors, using BSA and human serum gamma-globulin as substrates. Peptidases were detected by 1- and 2-D zymography in a polyacrylamide gel containing gelatin as substrate. Proteolytic activity was observed over a wide range of molecular masses equal to, or higher than, 45 kDa. To identify the peptidases, the extracellular proteins were digested with trypsin and analyzed by LC and MS. Seventeen peptides showing identity or similarity to predicted plant aspartic, cysteine, and serine peptidases have been identified. The extracellular localization of a cysteine peptidase aleurain homolog was also shown.

NtSCP1 from Tobacco Is an Extracellular Serine Carboxypeptidase III That Has an Impact on Cell Elongation

“…of a previous MS analysis that identified some of the peptides corresponding to the SCPIII sequence in the leaf intercellular fluid (Delannoy et al, 2008) and our own MS results for the extracellular medium of BY-2 cells (data not shown). Moreover, western blotting using anti-His antibodies showed that NtSCP1-6-His was found in the leaf intercellular fluid of transgenic plant lines (Fig.…”

“…cDNA Cloning of NtSCP1 and NtSCP2 from Tobacco Leaves Mass spectrometry (MS) analysis of tobacco leaf intercellular fluid resulted in the identification of tryptic fragments typical of SCPIII proteins (Delannoy et al, 2008). To clone the corresponding cDNA, degenerated SCPIII primers were designed based on the conserved upstream (MFYF/LF/LFESR) and downstream (VHDAGHMVPMDQPK) motifs of plant SCPIIIs in the MEROPS peptidase database (Supplemental Fig.…”

“…Whether these peptidases recognize one or several targets and whether their action is concerted with that of other exopeptidases or endopeptidases is unknown. Indeed, the extracellular medium has been shown to contain many peptidases (Delannoy et al, 2008), most of which have no known physiological role. Since cell elongation involves cell wall remodeling and cell turgor pressure, NtSCP1 might target any protein directly or indirectly involved in one of these processes.…”

“…A previous proteomics analysis of tobacco leaf intercellular fluid (Delannoy et al, 2008) identified a tryptic peptide that fitted the sequence of NtSCP1 or NtSCP2 (Supplemental Fig. S2).…”

“…Other extracellular SCPs have been identified in proteomic studies performed on the extracellular medium of Arabidopsis and pea (Pisum sativum) cells (Wen et al, 2007), but these have not been studied biochemically. Recently, we identified a type III SCP in a proteomic study of the extracellular space of tobacco (Nicotiana tabacum) leaves (Delannoy et al, 2008). Since no SCP type III (SCPIII) had been previously identified in the extracellular space, we cloned two SCP cDNAs, NtSCP1 and NtSCP2, characterized the former in more detail in terms of its expression pattern, subcellular localization, and enzyme activity, and found that transgenic plants overexpressing NtSCP1 show reduced cell elongation.…”