Despite strep-MBP-ROXY9 being partially oxidized, the protein was tested for its catalytic activity in vitro: The reduction capacity towards the artificial substrate HEDS and the deglutathionylation activity towards the protein substrate GAPDH, as well as the protein disulphide reduction activity towards insulin were assayed. As positive controls, the well characterized human glutaredoxin Grx1 [242,255–257] and the plant glutaredoxin GRXC2 from A. thaliana were chosen. The GRXC2 homolog in poplar was already characterized regarding its catalytic properties [77] and also for A. thaliana GRXC2, a catalytic activity was recently described [88].
Grx1 was expressed as a SUMO fusion in E. coli and purified via its strep-tag (Figure R12). The 27 kDa protein showed the same anomalous migration as already observed for strep-SUMO-ROXY9 during SDS-PAGE (Figure R5A). Since the purification necessitated the use of low-salt buffer, some contaminations with E. coli proteins remained in elution fractions 2 and 3 of strep-SUMO-Grx1. GRXC2 was fused to a strep-MBP-tag and expressed in E. coli, as well. The protein was purified via its MBP tag using an ÄKTA purifier (Figure R13). After switching to the elution buffer B, only two small peaks could be detected by measuring A280nm (Figure R13A). SDS-PAGE showed,
Figure R12. Purification of strep-SUMO-Grx1 from E. coli. Strep-MBP-Grx1 was expressed in E. coli BL21-star and purified using a StrepTactin gravitiy flow column. 12 % SDS-PAGE and Coomassie staining visualized the course of the purification. 10 µl of CE, FT, and
141 Figure R13. Purification of strep-MBP-GRXC2 from E. coli. Strep-MBP-GRXC2 was expressed in E. coli BL21-star and purified via its MBP tag using an ÄKTA purification system.
Purification was monitiored by following A280nm (A). Buffer B represents 1x MBP elution buffer with 50 mM NaCl. 20 µl of each fraction were separated by 10 % SDS-PAGE. The gels were stained with Coomassie (B). The arrow points at strep-MBP-GRXC2, whereas the asterisk marks the strep-MBP tag. Prestained PageRuler (Thermo Scientific) was used as a size standard.
mAU – milli absorption units, M – Marker, CE – crude extract, 1 to 33 – fraction number.
that the first peak (fraction 28) corresponded to almost pure MBP-GRXC2 in very low amounts. The second peak belonged to a mixture of strep-MBP-GRXC2 with strep-MBP tag (Figure R13B, fractions 29 and 30). For this reason, the concentration of strep-SUMO-Grx1 fraction E1 and strep-MBP-GRXC2 Fraction 28 was determined using Bradford solution. When other elution fractions were used, the concentration was estimated by loading a known volume of the protein preparation on an SDS gel together with different, defined amounts of BSA. Both proteins were dialysed to remove DTT from the purification and tested along with strep-MBP-ROXY9 for catalytic activity.
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First, the deglutathionylation and/or disulphide reduction activity of all three proteins towards HEDS was addressed (Figure R14A and B). For both, strep-SUMO-Grx1 and strep-MBP-GRXC2, 5, 25 and 50 nM protein were tested.
For strep-MBP-ROXY9, the ten-fold concentrations were used. As a negative control, a reaction without enzyme, but with glutathione was used. Reduction activity was visualized by coupling the glutaredoxin reaction to the NADPH-consuming reaction of GSSG reductase. As shown in Figure R14B, GSH alone led to a slow decline in the absorption at A340nm. Strep-SUMO-Grx1 and strep-MBP-GRXC2 did not differ from the no-enzyme control when they were used at concentrations of 5 nM. At 25 and 50 nM, however, a deglutathionylation activity could be detected by the stronger decrease in A340nm. Strep-MBP-ROXY9 was not different from the no-enzyme control even though it was used in higher concentrations than the two control proteins, suggesting that it is unable to reduce HEDS.
Next, the capacity of all three proteins to reduce the intermolecular disulfides in insulin was addressed (Figure R14A, C and D). The concentrations of the proteins used were the same as for the HEDS assay. As a control served a sample containing GSH but lacking a glutaredoxin. Precipitation of the insoluble B chain of bovine insulin was followed by the increase in A650nm. The negative control led to an increase in the absorption after 1.5 h, indicating that GSH alone caused reduction of insulin to a small extent (Figure R14D). Strep-SUMO-Grx1 and strep-MBP-GRXC2 were active at a concentration of 25 and 50 nM, but not or only weakly at a concentration of 5 nM (Figure R14C and D). Similarly, strep-MBP-ROXY9 used at a concentration of 50 nM did not reduce insulin to a higher extent than observed for the no-enzyme control.
However, at 250 and 500 nM, strep-MBP-ROXY9 even seemed to slow down the reduction rate observed for glutathione alone (Figure R14D).
Because we could only observe this so far unexplainable effect of strep-MBP-ROXY9 towards insulin and no activity towards HEDS, we decided to use a protein substrate to test deglutathionylation. For this reason, strep-SUMO-Grx1 and strep-MBP-ROXY9 were tested for their deglutathionylation capacity towards glutathionylated GAPDH. As outlined in Methods (page 103), this assay relies on the inactivation of GAPDH by glutathionylation of its catalytic cysteine residue (GAPDH-SG) and reactivation by deglutathionylation through glutaredoxins [37]. Deglutathionylation is indirectly determined via the activity of GAPDH, the reaction of which needs reduced nicotinamid adenine dinucleotide (NADH) [37,78]. Figure R14E displays the initial velocity of GAPDH and GAPDH-SG after different time points of incubation with or
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without glutaredoxin. As controls, non-glutathionylated GAPDH, glutathionylated GAPDH and GAPDH-SG treated with DTT were used. The deglutathionylation of GAPDH-SG caused by GSH alone was determined by omitting the enzyme in the reaction mix. Whereas non-glutathionylated GAPDH was active, GAPDH-SG was completely inactive in this assay.
Reactivation of GAPDH-SG with an excess of DTT restored GAPDH activity, though not to the same levels as observed for GAPDH. This might be explained by partial reduction or by protein losses caused by the preparation procedure of GAPDH-SG, as the GAPDH activity measured for the non-glutathionylated control is caused by the maximum possible GAPDH concentration within the experiment. Glutathione treatment led to a much smaller enhancement of GAPDH activity over time, indicating that it restored GAPDH activity though not as efficiently as DTT. Strep-SUMO-Grx1 at a concentration of 40 nM reactivated approximately twice as much GAPDH as glutathione. After removal of DTT by dialysis, strep-MBP-ROXY9, although used again at almost ten-fold higher concentrations than strep-SUMO-Grx1, exhibited a weak to no activity compared to glutathione.
Assuming that ROXY9 might only act on a subset of proteins, redox-sensitive GFP (roGFP) was selected as another target to test for deglutathionylation activity of strep-MBP-ROXY9. This experiment was performed by Lara Ostermann (INRES, Prof. Dr. Markus Schwarzländer, Bonn). During this experiment, roGFP is first oxidized (roGFPox) and then incubated with GSH or GSH and glutaredoxin. The redox state of roGFP determines its fluorescence emission intensity at 510 nm after excitation at 400 nm and 482 nm. Oxidation of the protein leads to an increase of its fluorescence emission after excitation at 400 nm, whereas reduction causes an increase in the fluorescence emission after excitation at 482 nm [244]. Thus, redox reactions can be followed by monitoring the ratio of F400nm/F482nm. Samples containing roGFPox showed a high fluorescence ratio, while samples containing roGFPred showed a low fluorescence ratio (Figure SR4). In this experiment, GRXC1 from Arabidopsis purified by the department for Chemical Signalling (INRES) in Bonn was used as a positive control. Strep-MBP-ROXY9 was dialysed against the reaction buffer, thereby removing DTT. This treatment leads to an oxidation of the protein (Figure R11, sample A). The control protein, GRXC1, was used in the oxidized state in which it was purified.
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Figure R14, part I. In vitro reductase activity of strep-MBP-ROXY9 when DTT is removed by dialysis. Different concentrations of strep-SUMO-Grx1, strep-MBP-GRXC2 and strep-MBP-ROXY9 (A) were analyzed in the HEDS (B) and the insulin assay (C and D) for reductase activity. The diagrams show the change in the absorption at 340 nm (HEDS) and 650 nm (insulin) over time. In case of the insulin assay, the measurement was continued after a break at 4.5 h (C) for another 4.5 h (D). Each reaction was prepared only once.
145 Figure R14, part II. In vitro reductase activity of strep-MBP-ROXY9 when DTT is removed by dialysis. (E) Strep-SUMO-Grx1 and strep-MBP-ROXY9 were compared in the GAPDH assay. For this, ca. 40 nM of strep-SUMO-Grx1 and ca.300 nM strep-MBP-ROXY9 were added together with GSH to the reaction mixtures containing maximally 9.6 µM glutathionylated GAPDH (GAPDH-SG). The GAPDH activity was assessed by withdrawing aliquots during the reaction and measuring the velocity of GAPDH (v(GAPDH)) in these samples. The initial velocity of GAPDH was plotted over the timepoints at which a sample was taken. Treatment of GAPDH-SG with DTT or GSH alone served as controls.
Additionally, a sample of untreated GAPDH-SG and 9.6 µM GAPDH were analyzed. All samples in the GAPDH assay were prepared only once.(F and G) strep-MBP-ROXY9 and N-terminally His-tagged GRXC1 were compared regarding the reduction of oxidized roGFP2.
Strep-MBP-ROXY9 and GRXC1 were used in an oxidized state. The indicated amounts of GRXC1 (F) and strep-MBP-ROXY9 (G) were incubated with or without GSH and oxidized roGFP. The reactions were started after 10 min of preincubation. The initial velocity of these reactions measured in the linear reaction phase between minutes 13 and 15 was plotted in the bar charts. Values represent mean ± SD.
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Figures R14F and G display the slope of the increasing reduction of roGFP for samples containing 0, 1, 2, or 5 µM of glutaredoxin, either with or without glutathione. The fluorescence changes over the entire experiments are shown in Figure SR4. GRXC1 (Figure R14F) led to a significant increase in the amount of reduced roGFP compared to the controls without glutathione and without enzyme, or with glutathione alone. However, the reduction of roGFP by GRXC1 depended on glutathione, as no activity was observed for samples containing enzyme, but lacking glutathione. In contrast, for strep-MBP-ROXY9, only the background activity caused by glutathione could be observed, even when protein was added to the reaction mixture (Figure R14G). Thus, while GRXC1 reduced roGFPox, strep-MBP-ROXY9 did not.