In vitro Glutamylation Inhibition of Ubiquitin Modification and Phosphoribosyl-Ubiquitin Ligation Mediated by Legionella pneumophila Effectors

Materials and Reagents

1. 1.7 ml Microtubes (Corning Incorporated, Axygen, catalog number: MCT-175-C )

2. 50 ml Centrifuge tubes (VWR, catalog number: 525-0637 )

3. Gloves (VWR, catalog number: 89038-270 )

4. Kim wipes (Kimberly-Clark Professional, catalog number: 34120 )

5. Pipette tips:

   10 μl XL Graduated Tips (USA Scientific, Tip One, catalog number: 1110-3700 )

   200 μl Graduated Quick Rack (Laboratory Products Sales, catalog number: 130430 )

   1,250 μl Pipette tips (Laboratory Product Sales, catalog number: L134770)

6. Recombinant proteins

   SidJ 89-853 truncation, SdeA 211-1152 truncation, human calmodulin 2, were expressed in Escherichia coli with an N-terminal 6XHis-SUMO tag and purified as described previously (Sulpizio et al., 2019). Rab33b (1-200) was also purified as proteins mentioned in (Sulpizio et al., 2019) and Ub was purified as described in Akturk et al. (2018). Final purified proteins were stored in buffer (20 mM Tris pH 7.5, 150 mM NaCl) without glycerol, aliquoted, flash-frozen, and stored at -80 °C.

7. β-Nicotinamide adenine dinucleotide sodium salt (NAD) (Sigma, catalog number: N0632-1G )

8. L-Glutamic Acid Monosodium Salt, Monohydrate (USB Corporation, catalog number: 16245 )

9. 2-Mercaptoethanol (Sigma, catalog number: M3148-100ML )

10. 30% Acrylamide/Bis solution 37.5:1 (Bio-Rad, catalog number: 1610158 )

11. Acetic acid, glacial (J.T. Baker, catalog number: 9508-06)

12. Adenosine 5’-triphosphate disodium salt hydrate (Sigma, catalog number: A2382-10G )

13. Ammonium persulfate (APS) (Amresco, catalog number: 0486-100G )

14. Brilliant Blue R-250 (Fisher, catalog number: BP101-50 )

15. Bromophenol Blue sodium salt (Fisher, catalog number: BP114-25 )

16. DL-Dithiothreitol (DTT) (Amresco, catalog number: M109-25g )

17. Ethanol 200-proof (Koptec, catalog number: V1001 )

18. Glycerol (Mallinckrodt Chemicals, catalog number: 5092-16 )

19. Glycine (VWR, catalog number: 0167-5KG )

20. Magnesium chloride, 6-hydrate (Mallinckrodt Chemicals, catalog number: 5958-04 )

21. Methanol (Fisher, catalog number: A454SK-4 )

22. N,N,N’,N’-tetramethylethylene-diamine (TEMED) (Bio-Rad, catalog number: 161-0800 )

23. Precision Plus Protein All Blue Standards Protein Ladder (Bio-Rad, catalog number: 161-0373 )

24. Sodium chloride (VWR, catalog number: 0241-10KG )

25. Sodium dodecyl sulfate (SDS) (VWR Life Sciences, catalog number: 0227-1KG )

26. Tris (VWR, catalog number: 0497-5KG )

27. Reaction Buffer (see Recipes)

28. MgCl₂ 1 M Solution (see Recipes)

29. ATP 100 mM pH 7.5 Solution (see Recipes)

30. Glutamic Acid 1 M Solution (see Recipes)

31. 10x SDS-PAGE Running Buffer (see Recipes)

32. 10x Native-PAGE Running Buffer (see Recipes)

33. SDS Sample Buffer (see Recipes)

34. Native Sample Buffer (see Recipes)

35. Coomassie Stain (see Recipes)

36. Coomassie Destaining Solution (see Recipes)

37. 12% SDS-PAGE Resolving Gel (see Recipes)

38. 4% SDS-PAGE Stacking Gel (see Recipes)

39. 8% Native PAGE Resolving Gel (see Recipes)

40. 3% Native PAGE Stacking Gel (see Recipes)

    Note: Products were stored as suggested by manufacture except where listed.

Equipment

1. -80 °C freezer (So-Low, model: PV85-21 )

2. Computer

3. Dry Bath (Benchmark, model: BSH1001 )

4. Fixed speed centrifuge (Benchmark, model: C1008-C )

5. Forceps

6. Gel electrophoresis apparatus (Bio-Rad, model: Mini-PROTEAN Tetra System )

7. Gel electrophoresis power supply (Bio-Rad, model: PowerPac Basic )

8. Gel imager (Bio-Rad, model: Chemidoc MP Imaging System )

9. Ice bucket

10. Labcoat (VWR, catalog number: 10141-306 )

11. Laboratory tape (VWR, catalog number: 89098-062 )

12. Microwave (Sharp, model: R230KW )

13. Pipettes (Gilson, model: Pipetman classic P2, P20, P200, P1000, catalog numbers: F144801 , F123600 , F123601 , F123602 )

14. Rocking shaker (Reliable Scientific, Inc., model: 55D 12 x 16 )

15. Sheet protectors (Clear file, Archival Plus 5x7 Print, catalog number: 370100B )

16. Vortex mixer 120V (Corning LSE, model: 6775 )

Procedure

1. SidJ in vitro Glutamylation Reaction

   1. Review the flowchart of the experimental outline of SidJ glutamylation and SdeA activity assays before beginning the procedure (Figure 1).

      
      

      
      Figure 1. Experimental outline for SidJ in vitro Glutamylation and SdeA Inhibition assay. A flow chart of the general experimental steps described in this procedure. The left branch is to assay the inhibition of SdeA ADP-ribosylation or Phospho-Ribosylation of Ub. The right branch is the experiments to assay PR-Ub ligation to substrates.

      
      

   2. Thaw recombinantly purified SidJ 89-853, SdeA Core (truncation 211-1152), calmodulin, ubiquitin, and optionally, Rab33b (1-200) on ice.

      Note: Rab33b 1-200 was used in this assay due to the prominence of a single PR-Ubiquitination band and increased protein stability (Jon Wasilko, Mao Lab, unpublished results). Full-length Rab33b may also be used instead.

   3. Preheat dry bath to 37 °C.

   4. Prepare stock solutions, on ice, listed in Table 1 by diluting in reaction buffer (20 mM Tris pH 7.5, 50 mM NaCl). Prepare NAD solution fresh and use glutamic acid and ATP stocks stored at -80 °C.

      
      

      Table 1. Stock solution concentrations for SidJ in vitro glutamylation and SdeA inhibition reaction
      

      

   5. Pipette the volumes of stock solution listed in SidJ glutamylation reaction row for a 25 μl reaction listed in Table 2 into a chilled 1.7 ml microcentrifuge tube on ice. Pipette ATP last to initiate the reaction. Immediately vortex, centrifuge briefly (~10 s max speed), and incubate the samples at 37 °C in a dry bath for 30 min.

      Note: It is important that samples are mixed thoroughly and centrifuged. SdeA is very active and the reaction mixture needs to be homogenous for maximal inhibition.

   6. Optional: Prepare a master mix for Step A5. Combine components contained in all reactions by pipetting stock solutions and 10 μl of reaction buffer per reaction. Prepare approximately 10% more reaction mix than needed for samples. The addition of reaction buffer to the master mix dilutes components to maintain protein stability. If a master mix is prepared, for each reaction, subtract the volumes of reaction components included in the mix and 10 μl of reaction buffer from the amount used in Table 2.

      
      

      Table 2. SidJ in vitro glutamylation and SdeA modification reaction components and concentrations
      

      

   7. Pipette remaining reaction components for the SdeA activity portion of the assay into the tube. If assaying ubiquitin modification, replace Rab33b with reaction buffer. Immediately vortex, centrifuge briefly (~10 s max speed), and incubate samples at 37 °C in a dry bath for 30 min.

      Note: A master mix containing SdeA modification components and 3 μl reaction buffer per sample can be prepared to minimize pipetting inaccuracies. A negative control excluding NAD would be beneficial for the visualization of the absence of SdeA activity.

   8. If assaying ubiquitin modification, separate each reaction into two tubes by pipetting 12.5 μl into another 1.7 ml microcentrifuge tube. Label tubes and pipette 3 μl of native sample buffer into one tube for native-PAGE analysis, and 3 μl of SDS sample buffer into the other for SDS-PAGE analysis and visualization of protein loading. Vortex to mix and centrifuge briefly (10 s at max speed). If only assaying PR-ubiquitin ligation activity, the reaction can be halted by the addition of 6 μl of SDS sample buffer.

      Note: Do not include SDS in native sample buffer, native-PAGE gels, or native-PAGE running buffer.




2. Detection of ubiquitin modification and PR-Ubiquitin ligation to Rab33b by gel electrophoresis

   1. For detection of ubiquitin modification, electrophorese 13 μl of the portion of each sample in native sample buffer using a native-PAGE gel and native-PAGE running buffer at 80 V. Once samples have migrated through stacking gel, increase the voltage to 120 V and electrophorese until dye front migrates 50-75% through the gel.

      Note: Native-PAGE is required to detect modification of ubiquitin. The use of cold native-PAGE running buffer, placement of gel apparatus on ice, and shortened electrophoresis distance may provide better separation and clarity of protein bands.

   2. For detection of PR-Ubiquitin ligation and visualization of protein loading for ubiquitin modification gel, electrophorese 2.5 μl of protein ladder and 13 μl of each reaction in SDS sample buffer using an SDS-PAGE gel (4% separating gel, 12% resolving gel) and SDS-PAGE running buffer at 80 V. Once samples have migrated through stacking gel, increase the voltage to 150 V and electrophorese until dye front reaches the bottom of the gel.

   3. Separate the gel from the casting glass and remove the stacking gel. Transfer the gel to a microwave-safe, plastic container with a lid. Pour Coomassie stain to cover gel and microwave briefly in a covered container, until boiling. Ensure not to inhale fumes when moving the container by maximizing distance from the container. Stain gel by rocking for a few hours, to overnight, at room temperature.

      Note: There was some difficulty visualizing the calmodulin band using Coomassie staining. Staining temperature and time may be decreased if calmodulin can be adequately stained.

   4. Discard stain and rinse with the destaining solution and incubate in the destaining solution for approximately 30 min to 1 h. Discard solution and repeat incubation. Repeat until protein bands are visible. If some background staining persists, allow longer water destaining in Step B5.

   5. Rehydrate gel and destain further by incubation in ddH₂O while rocking for 1-2 h. Remove water and repeat if necessary. The addition of a Kim wipe can assist in destaining and provide cleaner gel images.

   6. After the gel is rehydrated, transfer the gel to a sheet protector and image using the gel imager. Wiping dust and staining imperfections with Kim wipe may help obtain clearer images (Figure 2).

   7. Analyze results. The addition of phosphoribose or ADP-ribose to ubiquitin by SdeA causes a significant charge alteration relative to the overall size of the small ubiquitin protein. As a result, this alteration greatly shifts the electrophoretic mobility on a native gel, while this modification is not visualized if separating only by protein size as with SDS-PAGE. The inhibition of SdeA prevents the migration shift of ubiquitin (Figure 2A). This assay does not distinguish between the addition of ADP-ribose and further modification to PR-Ub. If assaying the PR-Ubiquitin ligation to Rab33b by SdeA, ligation is detected by incremental 8 kDa band shifts above the unmodified Rab33b band in the presence of NAD. This appearance of increased molecular weight bands corresponds to the attachment of one or multiple PR-Ub to Rab33b. Inhibition of SdeA’s PR-Ubiquitin ligase activity should decrease the intensity of the modified Rab33b compared to the uninhibited SdeA reaction (Figure 2B).

   
   

Figure 2. Reaction components required for SidJ mediated glutamylation and inhibition of SdeA. A. SidJ inhibition of the ability of SdeA to modify ubiquitin. SidJ glutamylation and SdeA modification were conducted with the reaction components and concentrations listed in Table 2, with Rab33b 1-200 replaced with reaction buffer. Reaction components were excluded as indicated. SidJ in vitro glutamylation assay was conducted for 30 min at 37 °C followed by SdeA modification for 30 min at 37 °C. Proteins were electrophoresed by native-PAGE and stained with Coomassie stain. B. SidJ inhibition of the ability of SdeA to PR-Ubiquitinate Rab33b 1-200. The reaction was conducted with concentrations listed in Table 2. SidJ in vitro glutamylation assay was conducted for 30 min at 37 °C followed by SdeA PR-Ubiquitination for 30 min at 37 °C. Proteins were electrophoresed by SDS-PAGE and the gel was stained with Coomassie stain. This figure is from the original research article (Sulpizio et al., 2019).

Recipes

1. Reaction Buffer

   50 mM Tris pH 7.5

   50 mM NaCl

   Stored at room temperature

2. MgCl₂ 1 M Solution

   Weigh 2.033 g of magnesium chloride, 6-hydrate and dilute to 10 ml with ddH₂O

   Store at room temperature

3. ATP 100 mM pH 7.5 Solution

   Weigh 551.14 mg adenosine 5′-triphosphate disodium salt hydrate and dissolve in 8 ml of ddH₂O

   Adjust the pH to 7.5 with 10 M sodium hydroxide and dilute to a final volume of 10 ml with ddH₂O

   Aliquot and store at -80 °C

4. Glutamic Acid 1 M Solution

   Weigh 0.936 g of L-glutamic acid monosodium salt, monohydrate and dilute to 5 ml with ddH₂O

   Aliquot and store at -80 °C or prepare fresh

5. 10x SDS-PAGE Running Buffer

   Store at room temperature and dilute 10-fold with ddH₂O for use

   Component	1 L
   Tris-Base	30 g
   Glycine	140 g
   SDS	10 g
   ddH2O	To 1 L

6. 10x Native-PAGE Running Buffer

   Store at room temperature and dilute 10-fold with ddH₂O for use

   Component	1 L
   Tris-Base	35 g
   Glycine	144 g

   ddH₂O         To 1 L

7. SDS Sample Buffer

   Store at room temperature, freeze aliquots -20 °C for extended storage

   Component             Concentration
   Bromophenol Blue     0.25% (w/v)
   DTT                                0.5 M
   Glycerol                         50% (v/v)
   SDS                                10% (w/v)
   2-Mercaptoethanol     10% (v/v)

8. 6x Native Sample Buffer

   Store at room temperature

   Component                50 ml
   ddH₂O                         35 ml
   Glycerol                       15 ml
   Bromophenol Blue    0.125 g

9. Coomassie Stain

   Store at room temperature

   Component                 500 ml
   Methanol                     225 ml
   ddH₂O                          225 ml
   Glacial Acetic Acid      50 ml
   Brilliant Blue R250      1.25 g

10. Coomassie Destaining Solution

    Store at room temperature

    Component                  1 L
    Ethanol                        450 ml
    ddH₂O                         450 ml
    Glacial Acetic Acid      100 ml

    
    

    SDS-PAGE Gel

11. 12% Resolving Gel

    Component                                                  8 gels                                    Final Conc.
    ddH₂O                                                           13.3 ml
    30% Acrylamide/Bis Solution                     16 ml                                       12%
    1.5 M Tris pH 8.8                                          10 ml                                        375 mM
    10% SDS                                                        400 μl                                        0.1%
    10% APS                                                        300 μl                                         0.075%
    TEMED                                                           24 μl                                           0.06%

12. 4% Stacking Gel

    Component                                                   8 gels                                     Final Conc.
    ddH₂O                                                            11.2 ml
    30% Acrylamide/Bis Solution                      2.16 ml                                     4.14%
    1.0 M Tris pH 6.8                                           2 ml                                          128 mM
    10% SDS                                                         160 μl                                        0.1%
    10% APS                                                          110 μl                                       0.07%
    TEMED                                                             16 μl                                           0.1%

    
    

    Native-PAGE Gel

13. 8% Resolving Gel

    Component                                                 20 ml (4 Gels)                            Final Conc.
    ddH₂O                                                               9.34 ml
    1.5 M Tris, pH 8.8                                             5 ml                                        375 mM
    30% Acrylamide/Bis Solution                         5.34 ml                                   8%
    10% APS                                                            100 μl                                      0.05%
    TEMED                                                               20 μl                                        0.1%

14. 3% Stacking Gel

    Component                                                 10 ml (4 Gels)                             Final Conc.
    ddH₂O                                                           8.32 ml
    1.0 M Tris, pH 6.8                                         500 μl                                          50 mM
    30% Acrylamide/Bis Solution                    1.02 ml                                         3%
    10% APS                                                        50 μl                                            0.05%
    TEMED                                                           10 μl                                            0.1%

Acknowledgments

This work was supported by National Institute of Health (NIH) grant 5R01GM116964 (to YM), the Cornell University Harry and Samuel Mann Outstanding Graduate Student Award (to AGS), and by the NIH under Ruth L Kirschstein National Research Service Award (6T32GM008267) from the NIGMS (to MEM).

   Protocol from original research article: Sulpizio, A., M. E. Minelli, M. Wan, P. D. Burrowes, X. Wu, E. J. Sanford, J.-H. Shin, B. C. Williams, M. L. Goldberg, M. B. Smolka, and Y. Mao (2019). "Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ." eLife 8: e51162.

Competing interests

The authors declare no competing interests.

References

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