The protease-associated domain and C-terminal extension are required for zymogen processing, sorting within the secretory pathway, and activity of tomato subtilase 3 (SlSBT3)

Abstract

A transgenic plant cell suspension culture was established as a versatile and efficient expression system for the subtilase SlSBT3 from tomato. The recombinant protease was purified to homogeneity from culture supernatants by fractionated ammonium sulfate precipitation, batch adsorption to cation exchange material, and anion exchange chromatography. Purified SlSBT3 was identified as a 79-kDa glycoprotein with both complex and paucimannosidic type glycan chains at Asn(177), Asn(203), Asn(376), Asn(697), and Asn(745). SlSBT3 was found to be a very stable enzyme, being fully active at 60 degrees C and showing highest activity at alkaline conditions with a maximum between pH 7.5 and 8.0. Substrate specificity of SlSBT3 was analyzed in detail, revealing a preference for Gln and Lys in the P(1) and P(2) positions of oligopeptide substrates, respectively. Similar to bacterial, yeast, and mammalian subtilases, SlSBT3 is synthesized as a preproenzyme, and processing of the prodomain in the endoplasmic reticulum is a prerequisite for passage through the secretory pathway. SlSBT3 S538A and S538C active site mutants accumulated intracellularly as unprocessed zymogens, indicating that prodomain cleavage occurs autocatalytically. The wild-type SlSBT3 protein failed to cleave the prodomain of the S538A mutant in trans, demonstrating that zymogen maturation is an intramolecular process. Distinguishing features of plant as compared with mammalian subtilases include the insertion of a large protease-associated domain between the His and Ser residues of the catalytic triad and the C-terminal extension to the catalytic domain. Both features were found to be required for SlSBT3 activity and, consequently, for prodomain processing and secretion.

FIGURE 1.

Primary structure of SlSBT3. The different domains of SlSBT3 are shown in a schematic representation of the SlSBT3 primary structure (A). The numbers indicate the amino acids of the catalytic triad (Asp, His, and Ser) and at the domain junctions. The entire amino acid sequence of the SlSBT3 zymogen is shown in B. The arrows indicate the processing sites of the signal peptide and the prodomain, respectively. The amino acids identified by N-terminal sequence analysis of purified SlSBT3 and the C-terminal SPI motif are shown in boldface type. Active site residues are marked by diamonds. Tryptic peptides identified by mass spectrometry are underlined, potential glycosylation sites are shown in boldface italic type, and those that were confirmed experimentally are double underlined. Five amino acids downstream from the prodomain processing site, the double arrow indicates the position where a Myc tag (EQKLISEEDL) was inserted in order to generate a tagged version of SlSBT3.

FIGURE 2.

Purification of SlSBT3 (A) and analysis of glycosylation (B). Recombinant SlSBT3 from cell suspension cultures was analyzed by SDS-PAGE (A, top) and on Western blots (A, bottom). The progress of SlSBT3 purification from cell culture supernatant was monitored after ammonium sulfate precipitation (ASP) and anion exchange chromatography (AEX). Four μg and 100 ng of purified SlSBT3 were loaded on the Coomassie Brilliant Blue-stained gel (top) and immunoblot (bottom), respectively. The size and position of marker proteins (Fermentas, St. Leon-Rot, Germany) are indicated (M). B, glycosylation of SlSBT3. 5, 10, 15, and 20 μg of purified SlSBT3 and RNase B as a positive control were separated by SDS-PAGE, and sugars were detected by periodic acid Schiff staining.

FIGURE 3.

Enzymatic properties of SlSBT3. Each experiment was performed in triplicate, using three independently purified batches of recombinant SlSBT3. A, pH optimum. SlSBT3 activity was assayed at different pH values in a tricomponent buffer system of constant ionic strength. B, heat stability. The assays were incubated for 20 min at the temperatures indicated and subsequently chilled on ice. Each reaction was started by the addition of substrate, and residual SlSBT3 activity was assayed at 25 °C. C, inhibitor profile. SlSBT3 was incubated for 20 min in assay buffer containing the different protease inhibitors. Inhibitor concentration is given in brackets in mm. Reactions were started by the addition of substrate. In A–C, activity is expressed as a percentage of the highest value or control, with 100% corresponding to 111 ± 13 pmol/min, 151 ± 36 pmol/min, and 143 ± 16 pmol/min in A, B, and C, respectively. D, steady state kinetic analysis. In three independent experiments, SlSBT3 activity (nmol/min) was assayed at substrate concentrations ranging from 2.5 to 200 μm. In order to derive the kinetic constants, the data were fitted to the Michaelis-Menten equation.

FIGURE 4.

Cleavage of synthetic peptides by SlSBT3. Synthetic peptides were incubated with recombinant SlSBT3, and the cleavage products were analyzed by MALDI-TOF mass spectrometry after 2 and 15 h, respectively. Sites that were efficiently processed after 2 h are indicated by heavy arrows; lighter arrows mark sites that were only partially processed even after 15 h of incubation. Gln residues in the P₁ position of cleaved sites are underlined.

FIGURE 5.

Processing and targeting of wild-type SlSBT3 and mutant variants after transient expression in N. benthamiana leaves. A, requirement of active site residues and the PA domain. Transiently expressed SlSBT3 (wt), its active site mutants with Ala or Cys replacing the active site Ser (S538A and S538C, respectively), and a mutant lacking the PA domain (SBT3ΔPA) were analyzed in total protein extracts (10 μg; top) and apoplastic (extracellular) washes (2.5 μg; bottom) and compared with the empty vector control (vector) and purified recombinant SlSBT3 (SBT3,C) on Western blots developed with a polyclonal SlSBT3 antiserum. The positions of the zymogen still retaining the prodomain (arrow) and the mature processed form of SlSBT3 (asterisk) are indicated. B, autocatalytic processing of SlSBT3. Total protein extracts from plants expressing only the Myc-tagged active site mutant of SLSBT3 (S538A-myc), co-expressing the mutant and wild-type SlSBT3 (S538A-myc + wt), and the empty vector control (vector) were analyzed on Western blots using antisera directed against SlSBT3 (top) and the Myc tag (bottom). The positions of the zymogen still retaining the prodomain (arrow) and the mature processed form of SlSBT3 (asterisk) are indicated. C, subcellular site of zymogen accumulation. Total protein extracts from plants expressing wild-type SlSBT3 (wt) and active site mutants (S528A and S538C), as well as apoplastic washes from plants expressing the wild-type enzyme (wash, wt), were treated (+) with Endo H (top) or PNGase F (bottom) and compared with untreated extracts (–) on Western blots using an SlSBT3 antiserum. The zymogens still retaining the prodomain (lanes marked S538A and S538C) are sensitive to endoglycosidase treatment (note size shift in treated as compared with untreated lanes). Mature SlSBT3 (lanes marked wt and the control lane containing purified recombinant SlSBT3 (SBT3,C)) is not sensitive to endoglycosidase treatment.

FIGURE 6.

C-terminal requirements for processing and targeting of SlSBT3. A range of C-terminally deleted SlSBT3 mutants was generated, which are shown schematically in A. The domain structure and the number of amino acids in the unprocessed precursors are indicated. The protein of 761 amino acids corresponds to wild-type SlSBT3. B, processing and secretion of C-terminal deletion mutants. Total protein extracts (20 μg of protein; top) and apoplastic washes (2.5 μg of protein; bottom) were prepared from N. benthamiana leaves transiently expressing the C-terminal variants of SlSBT3 and compared with the empty vector control (vector) and purified recombinant SlSBT3 (SBT3,C) on Western blots using an SlSBT3 antiserum. The arrow and asterisk indicate the zymogen still retaining the prodomain and the mature processed form of the SlSBT3 variants, respectively. The amino acid sequence at the extreme C terminus of different SBT3 mutants is shown in C, with the conserved SPI motif highlighted in boldface type.