Purification and characterization of serine proteases that exhibit caspase-like activity and are associated with programmed cell death in Avena sativa

Abstract

Victoria blight of Avena sativa (oat) is caused by the fungus Cochliobolus victoriae, which is pathogenic because of the production of the toxin victorin. The victorin-induced response in sensitive A. sativa has been characterized as a form of programmed cell death (PCD) and displays morphological and biochemical features similar to apoptosis, including chromatin condensation, DNA laddering, cell shrinkage, altered mitochondrial function, and ordered, substrate-specific proteolytic events. Victorin-induced proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is shown to be prevented by caspase-specific and general protease inhibitors. Evidence is presented for a signaling cascade leading to Rubisco proteolysis that involves multiple proteases. Furthermore, two proteases that are apparently involved in the Rubisco proteolytic cascade were purified and characterized. These proteases exhibit caspase specificity and display amino acid sequences homologous to plant subtilisin-like Ser proteases. The proteases are constitutively present in an active form and are relocalized to the extracellular fluid after induction of PCD by either victorin or heat shock. The role of the enzymes as processive proteases involved in a signal cascade during the PCD response is discussed.

Figure 1.

Inhibition of Rubisco Proteolysis with Protease Inhibitors. Leaf segments with epidermis removed were pretreated with 200 μM of the indicated inhibitor for 2 h then with 20 ng/mL of victorin for 4 h. Coomassie blue–stained 12% SDS-PAGE of total protein extract. Only the region containing Rubisco is shown.

Figure 2.

Characterization of Two Caspase-Like Proteolytic Activities. (A) Activity profile of fractions from a HIC column. The protease in activity A was assayed by incubating 50 μL of each fraction with 50 μL of 20 mM Mops, pH 7, with 20 μM substrate for 2 h. Activity B was assayed as described in Methods. RFU, relative fluorescence units. (B) Substrate profile of activity A. Activity A was assayed as described above for proteolytic activity with indicated substrates. RFU, relative fluorescence units. (C) Substrate profile of activity B. RFU, relative fluorescence units. (D) Inhibitor profile of activities A and B. Samples were pretreated with inhibitor (200 μM) for 2 h and then assayed for hydrolytic activity with Z-DEVD-AFC (activity A) or with Z-VAD-AFC (activity B). Expressed as percent inhibited of noninhibited control.

Figure 3.

The Caspase-Like Protease Is an 84-kD Ser Protease. (A) Reversibility of inhibition of the protease in activity B. Three distinct inhibitors all containing the VAD recognition motif were incubated at a concentration of 200 μM with activity B for 2 h. Samples were washed twice in buffer then reconstituted to original volume. After initial incubation and after each wash, aliquots were removed and assayed for Z-VAD-AFC hydrolytic activity. Irreversible inhibition of the protease was achieved only with the CMK-based inhibitor. (B) Biotin blot of the protease in activity B. Each lane contains a 30-μL sample of activity B from the HIC column. Samples were preincubated 2 h with a nonbiotinylated inhibitor (lanes 2 and 3) then incubated 2 h with biotin-YVAD-CMK (lanes 1 to 3). The caspase-like protease shows specific binding to biotin-YVAD-CMK because binding is competitive with Z-VAD-CMK, an inhibitor, but not Z-DEVD-CMK, which does not inhibit activity. Only the portion of the blot containing the 84-kD protein is shown.

Figure 4.

Release of the Saspase into the ECF after Victorin Treatment. (A) and (B) Leaves were infiltrated with 1 μg/mL of victorin (vic) or water (no victorin control), and the ECF was removed at the indicated time. (A) Z-VAD-AFC hydrolytic activity in the ECF. Activity expressed as relative fluorescence units. (B) Biotin blot of the ECF. Twenty microliters of each sample was incubated with 200 μM biotin-YVAD-CMK for 2 h before electrophoresis and blotting. (C) and (D) Leaves were preinfiltrated with 400 μM biotin-YVAD-CMK, preincubated 2 h, then infiltrated with 1 μg/mL of victorin mixed with 100 μM biotin-YVAD-CMK. (C) Biotin blot of the ECF. (D) Biotin blot of total protein extract. Note that the saspase is uniformly labeled in all samples including the water only/no victorin control (last lane).

Figure 5.

Heat Shock–Induced PCD in Victorin-Sensitive and -Insensitive A. sativa. Victorin-sensitive and -insensitive A. sativa leaves were infiltrated with water (lanes 1, 3, 4, and 6) or 1 μg/mL of victorin (lanes 2 and 5). Water-infiltrated leaves in lanes 3 and 6 were heat shocked for 60 min at 45°C and then incubated at 25°C for indicated times below. (A) Coomassie blue–stained SDS-polyacrylamide gel of total protein extracted 15 h after treatment. Rubisco cleavage is seen in sensitive leaves treated with victorin and sensitive and insensitive leaves that were heat shocked. Only the region containing Rubisco is shown. (B) 1.5% agarose gel of DNA extracted 15 h after treatment. (C) Biotin blot of ECF collected 4 h after treatment. Samples were incubated with 200 μM biotin-YVAD-CMK for 2 h before electrophoresis. (D) Z-VAD-AFC hydrolytic activity in the ECF. Activity expressed as relative fluorescence units.

Figure 6.

Inhibition of Heat Shock–Induced Rubisco Proteolysis. A. sativa leaves were preincubated with 400 μM of indicated inhibitor for 2 h and then heat shocked for 60 min at 45°C (sample in lane 1 did not undergo heat shock). Proteins were isolated 15 h after heat shock. Only the region containing Rubisco is shown.

Figure 7.

Anion Exchange Chromatography Separated Two Distinct Peaks of Saspase Activity. Silver stained gel of active fractions (20 μL loaded/lane) eluted off the anion exchange column. Z-VAD-AFC hydrolytic activity of each sample is indicated below the gel, expressed as relative fluorescence units. Fractions 42 to 52 (SAS-1) and 54 to 64 (SAS-2) were pooled for individual purification of SAS-1 and SAS-2 by size exclusion chromatography.

Figure 8.

Electrophoresis of SAS-1 and SAS-2 after Purification by Size Exclusion Chromatography. Silver stained gel of purified SAS-1 and SAS-2; 15 μL of active fraction was loaded per lane.

Figure 9.

Molecular Mass of both SAS-1 and SAS-2 Is ∼74.5 kD as Indicated by Mass Spectrometry. Spectrograph of purified SAS-1 (top spectrograph) and SAS-2 (bottom spectrograph) were individually analyzed by MOLDI-TOF mass spectrometry. First peak is the doubly charged ion, and the second peak is the singly charged ion.

Figure 10.

Peptide Maps of SAS-1 and SAS-2. Partial tryptic digest of SAS-1 and SAS-2 analyzed by MALDI-TOF mass spectrometry. Major peptide peaks with molecular mass differences between SAS-1 and SAS-2 are indicated by an arrow. m/z, mass-to-charge ratio.

Figure 11.

Effects of pH on Hydrolytic Activity of SAS-1 and SAS-2. Partially purified SAS-1 and SAS-2 (post-anion exchange chromatography) were assayed for hydrolytic activity at different pHs with the substrate Z-VAD-AFC. RFU, relative fluorescence units.

Figure 12.

Autoproteolysis of SAS-1 and SAS-2. Protein gel blot of purified SAS-1 and SAS-2 (200 ng each lane) boiled and not boiled before electrophoresis. Nonboiled enzymes underwent autoproteolysis with the appearance of lower molecular weight bands (a, b, c, and d).