Kinetic-based trapping by intervening sequence variants of the active sites of protein-disulfide isomerase identifies platelet protein substrates

Abstract

Thiol isomerases such as protein-disulfide isomerase (PDI) direct disulfide rearrangements required for proper folding of nascent proteins synthesized in the endoplasmic reticulum. Identifying PDI substrates is challenging because PDI catalyzes conformational changes that cannot be easily monitored (e.g. compared with proteolytic cleavage or amino acid phosphorylation); PDI has multiple substrates; and it can catalyze either oxidation, reduction, or isomerization of substrates. Kinetic-based substrate trapping wherein the active site motif CGHC is modified to CGHA to stabilize a PDI-substrate intermediate is effective in identifying some substrates. A limitation of this approach, however, is that it captures only substrates that are reduced by PDI, whereas many substrates are oxidized by PDI. By manipulating the highly conserved -GH- residues in the CGHC active site of PDI, we created PDI variants with a slowed reaction rate toward substrates. The prolonged intermediate state allowed us to identify protein substrates that have biased affinities for either oxidation or reduction by PDI. Because extracellular PDI is critical for thrombus formation but its extracellular substrates are not known, we evaluated the ability of these bidirectional trapping PDI variants to trap proteins released from platelets and on the platelet surface. Trapped proteins were identified by mass spectroscopy. Of the trapped substrate proteins identified by mass spectroscopy, five proteins, cathepsin G, glutaredoxin-1, thioredoxin, GP1b, and fibrinogen, showed a bias for oxidation, whereas annexin V, heparanase, ERp57, kallekrein-14, serpin B6, tetranectin, and collagen VI showed a bias for reduction. These bidirectional trapping variants will enable more comprehensive identification of thiol isomerase substrates and better elucidation of their cellular functions.

Keywords: disulfide; oxidase; platelet; protein disulfide isomerase; protein-protein interaction; reductase.

Figure 1.

PDI reduces and oxidizes substrate proteins. Shown is a reaction scheme diagramming the PDI active site transitioning between a reduced (left) and oxidized (right) state performing disulfide bond reduction (left to right) or oxidation (right to left) on a protein substrate. PDI active site (lower) and protein substrate (upper) are shown.

Figure 2.

Kinetic substrate trapping using either reduced or oxidized PDI variants. Shown are reaction schemes diagramming the kinetic trapping of either the wild-type enzyme, the CXXA variant, or the intervening sequence variants with substrate protein, starting from either the reduced (A) or oxidized (B) state. Bonds that are made or broken during catalysis are shown in red. t₀, the initial state; t₁ and t₂, intermediate states; t_trap, reaction state when stopped with NEM; t_final, protein-substrate complex, or lack thereof, isolated after IP.

Figure 3.

Effect on reductase activity of intervening sequence substitutions in the CXXC motif of PDI. A, PDI variants were tested for enzymatic activity using the insulin reductase assay. PDI was added to insulin in the presence of DTT, and turbidity was monitored at 650 nm for 1 h. B, variants that showed slower kinetics in the insulin reductase assay were tested for enzymatic activity using the di-eosin-GSSG assay. PDI was added to di-eosin-GSSG in the presence of DTT, and fluorescence was monitored for 10 min after excitation at 525 nm and recording emission at 545 nm. Wild-type PDI and the catalytically inactive AGHA PDI variant were used as positive and negative controls, respectively. Assays were performed in triplicate, and error bars represent standard deviation. ♦, wild-type PDI; ♢, AGHA-PDI; ▴, CAHC-PDI; ▵, CPHC-PDI; ●, CGPC-PDI; ○, CGRC-PDI; ■, CGAC-PDI; □, CGFC-PDI; , CGDC-PDI. RFU, relative fluorescence units.

Figure 4.

PDI variants retain activity following reversible oxidization and reduction. A, DTNB was used to measure free thiols on the PDI variants after reduction with DTT (gray bars) and oxidation with GSSG (white bars). Samples are normalized to the reduced wild-type PDI. B, PDI variants were tested for enzymatic activity after reduction (gray bars) and oxidation (white bars) using the di-eosin-GSSG assay. The catalytically inactive AGHA was used as a negative control. Assays were performed in triplicate, and error bars represent 3σ.

Figure 5.

Prereduced and preoxidized CGPC and CGRC form reduction-dependent complexes with substrates from activated washed platelets. Platelet releasates (A and B) and lysates (C and D) were prepared as described under “Experimental procedures.” Samples were then immunoprecipitated with anti-FLAG antibody, purposefully overloaded to detect PDI-substrate complexes, and separated by SDS-PAGE in either non-reducing (A and C) or reducing (B and D) conditions followed by immunoblotting using anti-FLAG antibody. The catalytically inactive AGHA variant was used as a control. Lane 1, prereduced CGPC-PDI; lane 2, prereduced CGRC-PDI; lane 3, prereduced AGHA-PDI; lane 4, preoxidized CGPC-PDI; lane 5, preoxidized CGRC-PDI; lane 6, preoxidized AGHA-PDI. Molecular mass standards are shown on the left and right.

Figure 6.

Reduction-dependent complexes are confirmed via separation with 2D electrophoresis. Platelet releasates and lysates were prepared as described. Proteins were first separated by SDS-PAGE under non-reducing conditions (left to right) and then separated again by SDS-PAGE under reducing conditions (top to bottom). Spots circled in red or green identify proteins specific for the CGRC variant. Spots boxed in blue or black squares identify proteins specific for the oxidized condition.

Figure 7.

Immunoblotting confirms complexed substrates identified via mass spectrometry. Platelet lysates and releasates were prepared as described. Samples were then immunoprecipitated with anti-FLAG antibody, purposely overloaded to detect PDI-substrate complexes, and separated by SDS-PAGE under either non-reducing (lanes 1–6) or reducing (lanes 7–12) conditions followed by immunoblotting onto PVDF membranes. Samples were visualized using both primary anti-FLAG antibody and primary antibody to either cathepsin G (A), heparanase (B), glutaredoxin-1 (C), or fibrin (D). Anti-FLAG signal is shown in green, target protein signal is displayed in red, and co-localization is displayed in yellow. The catalytically inactive AGHA variant was used as a control. Lanes 1 and 7, prereduced CGPC-PDI; lanes 2 and 8, prereduced CGRC-PDI; lanes 3 and 9, prereduced AGHA-PDI; lanes 4 and 10, preoxidized CGPC-PDI; lanes 5 and 11, preoxidized CGRC-PDI; lanes 6 and 12, preoxidized AGHA-PDI.

Figure 8.

The bias of the CGPC and CGRC PDI variants for forming complexes in their prereduced or preoxidized state. Shown is a graphical representation of the ratio of densitometry quantification of the target proteins identified for the CGPC variant (A) or the CGRC variant (B) in the prereduced or preoxidized samples. First, a target protein:PDI ratio was generated using the band intensities of both proteins from the oxidized or reduced samples (target:PDI variant). Then, a reduced:oxidized bias was generated using those values. Target proteins whose bias was toward the preoxidized state are oriented toward the left, and target proteins whose bias was toward the prereduced state are oriented toward the right. The black vertical bar represents the point of no bias, and the length of the bar represents the strength of the bias. Cath, cathepsin; Coll, collagen; Grx, glutaredoxin; Trx, thioredoxin; Fbgn, fibrinogen; KK, kallekrein.

Figure 9.

Effect of the oxidation state of PDI on cathepsin G activity in washed platelet releasate. Washed platelets were first incubated with either prereduced (gray) or preoxidized PDI (white) and then activated with 0.1 unit/ml thrombin. Platelet releasates were clarified with centrifugation and assayed for cathepsin G activity using a colorimetric probe monitored at 410 nm. Cathepsin G activities are shown as averages of triplicate experiments; error bars represent S.E. Paired t tests were used to determine significance: *, p < 0.05; **, p < 0.01. Red., reduced; Ox., oxidized; Cntrl, control; No Add., no additions.

Figure 10.

Kinetic substrate trapping using described PDI variants has potential to identify both reduction and oxidation substrates. Shown are reaction schemes diagramming the ideal reaction during kinetic trapping as well as a two-cycle kinetic trapping reaction. Wild type is shown for comparison. Green diagrams denote the hypothetical Protein X, blue diagrams denote the hypothetical Protein Y, and bonds that are made or broken during catalysis are shown in red. t₀, the initial state; t₁ and t₂, intermediate states; t_trap, reaction state when stopped with NEM; t_final, protein-substrate complex, or lack thereof, isolated after IP.