Radioactive Assay of in vitro Glutamylation Activity of the Legionella pneumophila Effector Protein SidJ

[Abstract] The Legionella effector protein SidJ has recently been identified to perform polyglutamylation on another Legionella effector, SdeA, ablating SdeA’s activity. SidJ is a kinase-like protein that requires the small eukaryotic protein calmodulin to perform glutamylation. Glutamylation is a relatively uncommon type of post-translational modification, where the amino group of a free glutamate amino acid is covalently linked to the γ -carboxyl group of a glutamate sidechain in a substrate protein. This protocol describes the SidJ glutamylation reaction using radioactive [U- 14 C] glutamate and its substrate SdeA, the separation of proteins by gel electrophoresis, preparation of gels for radioactive exposure, and relative quantification of glutamylation activity. This procedure is useful for the identification of substrates for glutamylation, characterization of substrate and glutamylase activities due to mutations, and identification of proteins with glutamylation activity. Some studies have assayed glutamylation with the use of [ 3 H] glutamate (Regnard et al. , 1998) and the use of the GT335 antibody (Wolff et al. , 1992). However, the use of [U- 14 C] glutamate requires a shorter radioactive exposure time with no dependence on antibody specificity.

independent manner. Using this binding ability, the structure of SidJ in complex with calmodulin was determined by X-ray crystallography. SidJ contains a kinase-like domain with structural homology to many conserved features found in kinases. This kinase domain is positioned in an active conformation through interactions with calmodulin. Based on these features of SidJ, reaction components were identified and used for in vitro glutamylation assays. Other assays have been developed using radioactive glutamate and gel extraction of modified substrates with liquid scintillation to detect modification (Black et al., 2019). Liquid scintillation and mass spectrometry may provide more precise quantification of the amount of modified substrate and for the number of glutamates attached to a substrate side-chain, respectively. The assay described in this protocol allows for visualization of activity by autoradiogram and relative quantitation of activity. This assay can be used to identify SidJ substrates and analyze the effect of point mutations on activity. In addition, this protocol may also be used to identify other proteins or pseudokinases that can function as glutamylases.   Note: When working with radioactive materials, ensure that all regulations are followed. Use proper PPE, maximize distance from the sample, and minimize exposure time.

Materials and Reagents
3. Warm water bath to 37 °C and chill centrifuge to 4 °C (if possible). Table 1 by diluting in reaction buffer (20 mM Tris pH 7.5, 50 mM NaCl). more reaction mix than needed for samples. The addition of reaction buffer for 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 3 μl of reaction buffer from the amount used in Table 2. 6. After the gel is rehydrated, transfer the gel to a sheet protector and image using a gel imager.

Prepare stock solutions, on ice, listed in
Wiping dust and staining imperfections with a Kim wipe may help obtain clearer images ( Figure   2A) 7. Cut a sheet of filter paper in half with scissors. Gently transfer gel to two stacked layers of filter paper, by touching one side of the gel to the paper, and then smoothly allowing it to lay flat.
Avoid bubbles and imperfections. Cover with a layer of plastic wrap and smooth out any imperfections. If there are lines in the plastic wrap, this may impact exposure efficiency.
8. Dry the gel using a vacuum gel dryer at 80 °C for 1 h or until the gel is completely dried. Ensure gel is dried before removing vacuum to prevent gel cracking. 9. While drying the gel, erase the image plate by exposing it on the lightbox for 30 min. 10. Wrap plastic wrap around the filter paper to prevent damage to the image plate. Tape the gel and two filter paper sheets to the bottom side of the exposure cassette. Avoid using thick plastic sheet protectors as they will reduce signal. 3. Click the "Start Scan" button and allow the instrument to scan the image plate. Once, the area containing your gel has been scanned acquisition can be stopped.
If a manual selection was chosen, ensure that the area containing the gel was accurately selected. Partial scanning of gels will prevent accurate comparison between portions of the gel scanned separately.

4.
A gel file will then be saved in the area chosen upon scanning. This can be saved to a flash drive and transported to a personal computer ( Figure 2B).

Data analysis
1. Open Fiji, or ImageJ, software. In the menu bar, select "File -> Open" and browse to the location of the .gel file.
If the gel is not horizontally level, select "Image -> Transform -> Rotate". Select the "Preview" checkbox and manually input a degree rotation and alter until gel preview is level. Then click "OK".
2. In the toolbar, select the rectangle selection tool. In the image window, click and drag to create a rectangular selection. This selection area should be large enough to fit the largest band. Then move this selection to cover the first band you would like to measure. In the menu bar, click "Analyze -> Measure." A Results pop up window will appear with intensity data. Click in the center of this rectangle and drag it to surround the next band to be analyzed and measure intensity. Repeat process, recording which measurements correspond to which samples until the intensity of all bands has been measured. Measure the intensity of a background selection, ideally on the area where the gel was exposed but no samples were electrophoresed. All background measurement from all samples. Relative intensities can be compared by dividing one sample's total intensity by another sample's total intensity. Calculation of relative intensities should only be made for samples exposed on the same image plate, and preferably electrophoresed within the same gel. For example, the intensity of protein mutants can be divided by the wild type total radiographic intensity to ascertain the effect of these mutants on activity. Removal or alteration of reaction components can also be compared.