S ITE - DIRECTED MUTAGENESIS OF CYSTEINES IN D ICTYOSTELIUM DISCOIDEUM

The results described above suggested that calcineurin inhibition by PAO and H2O2

involves oxidation of the protein cysteines. Identification of calcineurin cysteine residues sensitive to oxidation would help elucidate the mechanism, by which redox processes regulate calcineurin activity. X-ray structure of calcineurin provides the basis for assessment of cysteine residues most likely to be involved in oxidative inactivation. Bovine calcineurin A subunit has 12 cysteines, and the B subunit –2 cysteines. Previous studies on recombinant Dictyostelium discoideum calcineurin A showed that this protein is also sensitive to PAO and H2O2 [Hellstern, 1997], thus, the B subunit is unlikely to be the target of oxidative inactivation. Sequence alignment of bovine and Dictyostelium discoideum calcineurins reveals nine cysteine residues conserved between both species. The calculation of all distances between each possible pair of cysteine residues resulted in two pairs having the shortest inter-cysteine distances: Cys228-Cys256 (4.6 Å) and Cys166-Cys178 (5 Å). The Cys178 is not present in the enzyme from Dictyostelium discoideum. Therefore, only one pair of closely spaced cysteines able to interact with PAO is left in Dictyostelium discoideum calcineurin A, namely Cys278 and Cys305 (Dictyostelium discoideum nomenclature). To investigate the role of Cys278 and Cys305 in the interaction of calcineurin with oxidants site-directed mutagenesis of these cysteines to alanines was performed according to QuickChange protocol (Stratagene). Both single mutations as well as a double mutation were introduced into plasmid pQE30 CNA ∆N-terminus containing a 6His-tagged N-terminally truncated variant (lacking aminoacids 1-48) of Dictyostelium discoideum calcineurin A. The resulting mutations were confirmed by the restriction analysis of plasmid DNA as shown on Fig. 3.2.1.

Figure 3.2.1. Restriction analysis of wild type and C278A and C305A mutants of Dictyostelium discoideum calcineurin A. Plasmid mini-preps were treated with Ban I (lanes 1-3) or Eae I (lanes 4-6) restriction endonucleases and subjected to electrophoresis on 0.7% agarose gel. Lanes 1,4 – C305A; lanes 2,5 – C278A; lanes 3,6 – wild-type.

Lane 7 – undigested wild-type plasmid; lane 8 – DNA ladder.

Additionally, the correctness of all introduced mutations was confirmed by sequencing. The plasmids were then transformed into M15 E.coli strain, and the recombinant proteins expressed and purified to homogenity using FPLC on Ni-NTA agarose. A typical purification profile is shown on Fig. 3.2.2. A significant portion of calcineurin remained in the cell pellet (lane 6 on Fig 3.2.2); however, the amount remaining in the supernatant was satisfactory for further purification. The yields of recombinant proteins were generally 1-1.5 mg from 1 l culture medium.

Figure 3.2.2. Purification of the double mutant C278A/C305A. Proteins were separated by SDS-PAGE on 8% polyacrylamide gel and stained with Coomassie Blue. Lane 1 – lysate from non-induced culture; lanes 2-5 - lysates from cultures 1, 2, 3 and 4 hours after induction with 1 mM IPTG, respectively; lane 6 - the supernatant after French press; lane 7 – the pellet after French Press; lane 8 – flowthrough of the Ni-NTA column; lane 9 - wash fraction of the Ni-NTA column; lane 10 – elution fraction of the Ni-NTA column.

The specific activity of purified recombinant calcineurin A (wild type and mutants) towards pNPP was in the range of 100-400 Units/mg, i.e., significantly lower than that of the bovine calcineurin. The addition of calcineurin B did not significantly increase the activity towards pNPP, in agreement with previous data [Aichem, 2000]. Attempts to reconstitute and purify the holoenzyme were unsuccessful.

A B

C

3.2.2. REDOX-SENSITIVITY OF WILD-TYPE AND MUTATED PROTEINS.

Then the sensitivity of the wild type and mutant enzymes towards PAO and hydrogen peroxide were tested. Fig. 3.2.3. shows the concentration dependence of PAO inhibition of pNPP phosphatase activity of recombinant proteins.

Figure 3.2.3. Inhibition of recombinant Dictyostelium discoideum calcineurin A by PAO. pNPP phosphatase activity of wild type and mutant recombinant proteins was assayed in the presence of different PAO concentrations. Data show the activity 20 min after PAO addition. A.

Comparison of wild type and C278A mutant. B.

Comparison of wild type and C305A mutant. C.

Comparison of wild type and C278A/C305A mutant.

PAO inhibited recombinant proteins with IC50 ~20 µM. No significant difference in concentration dependence was observed between the wild type and either of the mutant proteins. Therefore, the inhibitory effect of PAO appears not to be mediated via interaction with Cys278 or Cys305 of Dictyostelium discoideum calcineurin A.

The effect of 30 min pre-incubation with 1 mM H2O2 on the activity of recombinant proteins was also investigated. A summary of the obtained results is shown on Fig. 3.2.4.

H2O2 inhibited Dictyostelium discoideum calcineurin A with IC50 ~0.2-0.5 mM. Similar to PAO inhibition, no significant difference between oxidative sensitivity of wild type and any of the mutant proteins was observed. It should be noted that, in contrast to bovine calcineurin, recombinant Dictyostelium discoideum proteins rapidly lost their activities in the absence of TCEP. Although this difference could partly be explained by longer pre-incubation times in

A B

C

case of recombinant proteins, one could also suggest that isolated calcineurin A subunit has an increased sensitivity to oxidation, probably via exposure of the residues otherwise hidden by calcineurin B subunit. Taken together, the results of H2O2 inhibition experiments confirm that redox sensitivity of Dictyostelium discoideum calcineurin is not mediated by cysteines 278 and 305, and probably their homologues Cys228 and Cys256 in bovine calcineurin.

Figure 3.2.4. Inhibition of recombinant Dictyostelium discoideum calcineurin A by H₂O₂. pNPP phosphatase activity of wild type and mutant recombinant proteins was assayed after 30 min pre-incubation with different H2O2 concentrations. A.

Comparison of wild type and C278A mutant. B. Comparison of wild type and C305A mutant. C. Comparison of wild type and C278A/C305A mutant.

3.2.3. REDOX SENSITITIVITY OF CYSTEINE-TO-ALANINE MUTANTS OF RAT CALCINEURIN A.

The sensitivity of C278A and C305A mutants to PAO and H2O2 leaves only seven cysteines on calcineurin A subunit able to participate in inactivation of the enzyme by oxidants. Recently, several cysteine-to-alanine mutants of rat calcineurin were obtained by Rusnak and associates [Reiter et al., 1999]. The mutations included C166A, C197A, and C88A/C197A substitutions. We obtained the possibility to investigate the redox sensitivity of the corresponding recombinant proteins. Plasmids encoding wild type and mutated rat calcineurin A in a pT7-7 vector were transformed into E.coli strain BL21(DE3). Expressed

proteins were reconstituted after lysis with rat calcineurin B-containing bacterial lysate and partly purified on calmodulin sepharose. PAO sensitivity of recombinant proteins was tested in the pNPP phosphatase assay. Neither of the mutant proteins showed PAO sensitivity different from the wild-type protein (Andrea Höngen, Diplom work, Universität Konstanz).

3.3. INHIBITION OF CALCINEURIN BY SUPEROXIDE IN TISSUE AND CELL HOMOGENATES.