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Mar 2020

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Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography
使用薄层和离子交换色谱评估二腺苷酸环化酶和c-di-AMP-磷酸二酯酶的活性   

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Abstract

All living cells use cyclic nucleotides as second messengers for signal sensing and transduction. Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is primarily involved in the control of bacterial and euryarcheal osmoadaptation and is produced by diadenylate cyclases from two molecules of ATP. Specific phosphodiesterases hydrolyze c-di-AMP to the linear phosphoadenylate adenosine 5′-pApA or to AMP. Different methods including high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC) and ion exchange chromatography (IEX) can be used to determine activities of c-di-AMP-synthesizing and degrading enzymes. Here, we describe in detail the TLC and IEX methods adapted for characterization of the diadenylate cyclase DisA and the phosphodiesterase AtaC from Streptomyces venezuelae. TLC allows quick and easy separation of radioactive-labeled substrates and products, while IEX avoids utilization of potentially hazardous radioactive substrates and can be used as a good substitute if an HPLC system is not available. Unlike in TLC assays, samples cannot be analyzed in parallel by using the IEX assay, thus it is more time consuming.

Keywords: c-di-AMP (环二腺苷酸), Diadenylate cyclase (二腺苷酸环化酶), DAC (DAC), DisA (DisA), Phosphodiesterase (磷酸二酯酶), PDE (PDE), AtaC (AtaC), Enzymatic assay (酶法测定)

Background

Cyclic nucleotide second messengers are key molecules in prokaryotic and eukaryotic signaling pathways. Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is a bacterial second messenger with many important functions, such as regulation of osmolyte homeostasis, cell wall metabolism, biofilm formation, DNA integrity, sporulation, virulence and growth (Fahmi et al., 2017). It is of particular interest as it is present in many human pathogens, such as Staphylococcus aureus, Mycobacterium tuberculosis and Streptococcus pneumoniae and was found to be essential for most species under normal growth conditions (Woodward et al., 2010; Corrigan et al., 2011; Luo and Helmann, 2012; Mehne et al., 2013; Whiteley et al., 2015). Intriguingly, also accumulation of c-di-AMP leads to growth defects (Bai et al., 2013; Mehne et al., 2013; Latoscha et al., 2020). Thus, the levels of this second messenger have to be tightly regulated. c-di-AMP is synthesized from two molecules of ATP by diadenylate cyclases (DAC) and degraded by specific phosphodiesterases (PDE) to 5′-pApA and/or two AMP molecules (Figure 1) (Fahmi et al., 2017; Yin et al., 2020).



Figure 1. Synthesis and degradation of c-di-AMP in S. venezuelae. The diadenylate cyclase DisA uses magnesium ions (Mg2+) as cofactors to synthesize c-di-AMP from two ATP molecules releasing two pyrophosphates (PPi) as a byproduct (Witte et al., 2008). c-di-AMP is degraded to 5′-pApA and two AMP molecules by the phosphodiesterase AtaC, which requires manganese ions (Mn2+) as cofactors (Latoscha et al., 2020) .


Synthesis of c-di-AMP by DAC domains was identified in five different protein types, termed DisA, CdaA, CdaS, CdaM and CdaZ. Most bacteria contain either DisA or CdaA for c-di-AMP synthesis, while some species, exclusively belonging to the genus Bacillus possess DisA, CdaA and CdaS in parallel (Commichau et al., 2019). Enzymatic activity of these proteins can be affected by different conditions. The most widespread DAC, CdaA, is regulated by the extracytoplasmic regulator CdaR and the phosphoglucosamine mutase GlmM (Tosi et al., 2019; Gibhardt et al., 2020). In contrast, DisA synthesizes c-di-AMP constitutively unless it binds branched or damaged DNA (Witte et al., 2008), which results in a sporulation delay (Bejerano-Sagie et al., 2006; Oppenheimer-Shaanan et al., 2011). Thus, the synthesis of c-di-AMP can be affected by many different environmental conditions, which are still not understood in detail. For c-di-AMP degradation, two major classes of specific PDEs were identified so far, referred to as DHH-type and HD-type PDEs, which are termed according to the conserved catalytic amino acid motifs Asp-His-His and His-Asp in their respective active sites. DHH-type PDEs are further divided into multidomain membrane-coupled proteins like GdpP (Pde1) and soluble standalone catalytic domain proteins of the DhhP-type (Pde2) (Rao et al., 2010; Bai et al., 2013; Ye et al., 2014). The HD-type PDE is a membrane-coupled member of the 7TMR-HD family (7 transmembrane domain receptor with HD domain), termed PgpH (Gundlach et al., 2015; Huynh et al., 2015). Both GdpP and PgpH exclusively hydrolyze c-di-AMP into 5′-pApA, which is further degraded into AMP by DhhP or nano-RNases, such as NrnA. DhhP PDEs often occur in parallel to GdpP or PgpH in one species and were shown to be responsible for the second degradation step from 5′-pApA to AMP (Bowman et al., 2016; Drexler et al., 2017; Konno et al., 2018). In contrast, some species, such as M. tuberculosis and Mycobacterium smegmatis only contain DhhP homologs, which were shown to hydrolyze both c-di-AMP and 5′-pApA (Tang et al., 2015; He et al., 2016). However, many species from the phylum Actinobacteria, including streptomycetes, do not possess any of the major c-di-AMP-specific PDE classes (Corrigan and Gründling, 2013; Yin et al., 2020). Instead, Streptomyces venezuelae utilizes an alternative PDE for c-di-AMP degradation. In our recent study, we applied different biochemical approaches to characterize DisA, the sole DAC domain protein encoded in Actinobacteria, as well as AtaC (Actinobacterial PDE targeting c-di-AMP), which is the founding member of a new family of c-di-AMP-specific PDEs and is mainly present in Actinobacteria (Latoscha et al., 2020).


For an accurate characterization of proteins in this signaling pathway, robust and sensitive assays for substrate specificity are required. In the present protocol, we describe two optimized methods for the analysis of c-di-AMP synthesis or degradation: the thin-layer chromatography assay (TLC) of enzymatic assays with radiolabeled nucleotides and the ion exchange chromatography assay (IEX).


The TLC assay was established initially to study the activities of Caulobacter crescentus diguanylate cyclase (DGC) PleD and c-di-GMP-specific PDE CC3396 (Paul et al., 2004; Christen et al., 2005) based on a method published by Ross et al. (1987). Detailed protocols on DGC and c-di-GMP-specific PDE radioactive assays coupled with TLC were published recently (Kazmierczak, 2017a and 2017b). In the last years, enzymatic assays with radiolabeled nucleotides coupled with TLC have been successfully used for characterization of c-di-GMP-specific enzymes from Gram-negative and Gram-positive bacteria (Kazmierczak et al., 2006; Bordeleau et al., 2011; Lindenberg et al., 2013; Al-Bassam et al., 2018) as well as of the DAC DisA from Bacillus subtilis and Thermotoga maritima (Witte et al., 2008; Torres et al., 2019). In contrast to other methods like high-performance liquid chromatography (HPLC), the TLC assay allows separation of substrate and products from multiple reactions at the same time. Moreover, a direct conclusion about substrate specificity can be drawn if the reaction was supplemented with cold nucleotides as competitors for enzymatic activity (Paul et al., 2004; Christen et al., 2005; Lindenberg et al., 2013). If required, a quantification of radioactive product formation can be performed since only radiolabeled nucleotides, but not the unlabeled competitors, are visualized after development of the TLC (Ross et al., 1987; Paul et al., 2004; Christen et al., 2005). In our recent study, we used the TLC assay to analyze both synthesis of c-di-AMP by S. venezuelae DisA and specificity of c-di-AMP degradation by AtaC (Latoscha et al., 2020).


IEX was used as an independent method to further characterize the activity of AtaC (Latoscha et al., 2020). In this assay, negatively charged molecules (e.g., nucleotides) bind to a positively charged column material and can be eluted by a salt gradient. Depending on their binding affinity to the column, the molecules elute at different salt concentrations and can be separated. The assay is of particular interest if other chromatography systems for small molecules, such as reverse-phase HPLC (RP-HPLC) are not available. Cyclic dinucleotides and their related reaction intermediates 5’-pNpN or NMP were first separated by ion exchange chromatography by Li et al. (2013) to analyze the reaction products of cyclic GMP-AMP synthase (cGAS). The method was further used by Luecke et al. (2017) and the respective assay was described in detail by Holleufer and Hartmann (2018). The method was adapted for the characterization of a PDE in the degradation pathway of c-di-AMP (Drexler et al., 2017) and was slightly amended for the present assay for the characterization of AtaC (Latoscha et al., 2020). In principle, this IEX assay can be used to analyze the activity of most cyclic dinucleotide synthesizing or degrading enzymes. However, a RP-HPLC system provides a faster and more precise separation of the samples and if it is available, then RP-HPLC would be the preferred chromatographic method to use, as described in several reports (Oppenheimer-Shaanan et al., 2011; Bai et al., 2012; Witte et al., 2013; Huynh et al., 2015; Tang et al., 2015).


Materials and Reagents

  1. Material for buffer exchange (see Notes)

    1. Locking clips, 45 mm (Carl Roth, catalog number: H277.1)

    2. Dialysis membrane Spectra/Por 7 MWCO 10,000, 45 mm (Carl Roth, catalog number: E872.1)

    3. Glass pipettes: 5, 10, 20 and 25 ml (Labdirect, catalog numbers: 021.01.005, 355.050.110, 355.050.120, 355.050.125)

    4. Pipette tips (Sarstedt, catalog numbers: 70.760.012, 70.762, 70.3020 and Biozym, catalog number: VZ0001X)

    5. 1.5 ml reaction tubes (Sarstedt, catalog number: 72.690.001)

    6. Double-distilled water (ddH2O) (Kerndl, catalog number: 22501)

    7. Magnesium chloride hexahydrate (MgCl2) (Carl Roth, catalog number: 2189.1)

    8. Manganese (II) chloride tetrahydrate (MnCl2) (Carl Roth, catalog number: T881.1)

    9. Sodium chloride (NaCl) (VWR, catalog number: 27810.295DB)

    10. Tris-base (Carl Roth, catalog number: 4855.2)

    11. Glycerol (Carl Roth, catalog number: 3783.2)

    12. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250)


  2. Material for protein concentration determination (see Notes)

    1. 1.5 ml reaction tubes (Sarstedt, catalog number: 72.690.001)

    2. Spacer plates and short plates (Bio-Rad, catalog numbers: 1653310 and 1653308)

    3. Pipette tips (Sarstedt, catalog numbers: 70.760.012, 70.762, 70.3020 and Biozym, catalog number: VZ0001X)

    4. Tris-base (Carl Roth, catalog number: 4855.2)

    5. Glycine (Carl Roth, catalog number: 0079.4)

    6. Colored Prestained Marker (NEB, catalog number: P7719S)

    7. LMW marker (GE Healthcare, catalog number: 17-0446-01)

    8. Double-distilled water (ddH2O) (Kerndl, catalog number: 22501)

    9. Rotiphorese (Carl Roth, catalog number: 3029.1)

    10. Ammonium peroxydisulphate (Carl Roth, catalog number: 9178.1)

    11. TEMED (Carl Roth, catalog number: 2367.3)

    12. Glycerol (Carl Roth, catalog number: 3783.2)

    13. Sodium lauryl sulphate (Carl Roth, catalog number: 4360.2)

    14. Bromophenol blue (Carl Roth, catalog number: A512.1)

    15. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250)

    16. 2-propanol (Carl Roth, catalog number: 6752.5)

    17. Acetic acid (Carl Roth, catalog number: 6755.1)

    18. Coomassie blue R 250 (Carl Roth, catalog number: 3862.2)


  3. Thin-layer chromatography (TLC) of enzymatic assays with radiolabeled nucleotides

    1. Pipette tips (Sarstedt, catalog number: 70.760.012 and Biozym, catalog number: VZ0001X)

    2. Pipette filter tips (Sarstedt, catalog number: 70.1116.210)

    3. 1.5 ml reaction tubes (Sarstedt, catalog number: 72.690.001)

    4. Thin-layer plate (Macherey-Nagel, POLYGRAM CEL 300 PEI, catalog number: 801053)

    5. Phosphor imaging plate (Fujifilm, BAS-IP SR 2025)

    6. Plastic wrap (any type obtained from a grocery store)

    7. Exposure cassette (GE Healthcare, catalog number: 63-0035-44)

    8. Purified DisA from S. venezuelae or B. subtilis (Witte et al., 2008; Latoscha et al., 2020)

    9. Purified inactive DisA variant (DisAD86A) from S. venezuelae (Latoscha et al., 2020)

    10. Purified AtaC from S. venezuelae (Latoscha et al., 2020)

    11. 9.25 MBq [α-32P]-ATP (Hartmann Analytik, catalog number: FP-207)

    12. 9.25 MBq [32P]-c-di-AMP (Hartmann Analytik, catalog number: FP-C517; a purified c-di-AMP-producing enzyme, e.g., Bacillus subtilis DisA, must be provided to the company for [32P]-c-di-AMP synthesis upon request)

    13. For phosphodiesterase competition assays:

      1. c-di-AMP (BioLog, catalog number: C 088)

      2. c-di-GMP (BioLog, catalog number: C 057)

      3. cAMP (Sigma-Aldrich, catalog number: A6885)

    14. Magnesium chloride hexahydrate (MgCl2) (Carl Roth, catalog number: 2189.1)

    15. Manganese (II) chloride tetrahydrate (MnCl2) (Carl Roth, catalog number: T881.1)

    16. Sodium chloride (NaCl) (VWR, catalog number: 27810.295DB)

    17. Tris-base (Carl Roth, catalog number: 4855.2)

    18. Hydrochloric acid (Carl Roth, catalog number: 4625.2)

    19. Glycerol (Carl Roth, catalog number: 3783.2)

    20. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250)

    21. Double-distilled water (ddH2O) (Kerndl, catalog number: 22501)

    22. 0.5 M EDTA, pH 8 (PanReac AppliChem, catalog number: A4892)

    23. Ammonium sulfate ((NH4)2SO4) (VWR, catalog number: 21333.296DB)

    24. Monopotassium phosphate (KH2PO4) (Carl Roth, catalog number: P018.2)

    25. DisA cyclase buffer (see Recipes)

    26. PDE reaction buffer (see Recipes)

    27. TLC running buffer (see Recipes)


  4. IEX activity assay

    1. Pipette tips (Sarstedt, catalog numbers: 70.1130.600 and 70.760.502)

    2. 1.5 ml reaction tubes (Sarstedt, catalog number: 72.690.001)

    3. Microcon-30kDa Centrifugal Filter Unit (Merck Millipore, catalog number: MRCF0R030)

    4. Purified AtaC from S. venezuelae (Latoscha et al., 2020)

    5. Purified TmPDE from T. maritima (Drexler et al., 2017)

    6. c-di-AMP (BioLog, catalog number: C 088)

    7. Manganese (II) chloride tetrahydrate (MnCl2) (Carl Roth, catalog number: T881.3)

    8. Sodium chloride (NaCl) (Merck Millipore, catalog number: 1.06404.5000)

    9. Tris-base (Carl Roth, catalog number: 4855.2)

    10. Hydrochloric acid, 25% (VWR, catalog number: 20257.296)

    11. 10x AtaC reaction buffer (see Recipes)

    12. IEX Running buffer A (see Recipes)

    13. IEX Running buffer B (see Recipes)

Equipment

  1. Equipment for buffer exchange (see Notes)

    1. Measuring beaker, 5,000 ml (Carl Roth, catalog number: 0780.1)

    2. Magnetic bar (Carl Roth, catalog number: C267.1)

    3. Pipetting aid macro (Carl Roth, catalog number: X478.1)

    4. Micropipettes (P20, P200, P1000)

    5. Magnetic stirrer (Heidolph, model: MR 2000)

    6. Centrifuge (Eppendorf, model: 5427 R, catalog number: 5409000010)

    7. 4 °C room


  2. Equipment for protein concentration determination of your choice (see Notes)

    1. Mini-PROTEAN Tetra Cell (Bio-Rad, catalog number: 1658000EDU)

    2. Heat block (Labnet International, model: AccuBlockTM Digital Dry Bath, catalog number: D1302-230V)

    3. Centrifuge (Eppendorf, model: 5427 R, catalog number: 5409000010)

    4. Power supply (Bio-Rad, catalog number: 1645050)

    5. Imager with CCD camera (GE Healthcare, model: ImageQuant LAS 4000)

    6. Micropipette (P2, P10, P20)


  3. TLC of enzymatic assays with radiolabeled nucleotides

    1. Micropipettes (P2, P10, P20)

    2. Heat block (Labnet International, model: AccuBlockTM Digital Dry Bath, catalog number: D1302-230V)

    3. Centrifuge (Eppendorf, model: 5427 R, catalog number: 5409000010)

    4. Contamination monitor (Berthold, model: LB 122)

    5. Biomolecular imager (GE Healthcare, model: Typhoon FLA 7000, catalog number: 28-9558-09)

    6. Image eraser (Molecular Dynamics, model: 410A)

    7. Acrylic Benchtop Beta Radiation Shield (ThermoFisher Scientific, catalog number: 6700-2418)

    8. TLC chamber (Fisher Scientific, catalog number: 06-815-187)

    9. Fume hood


  4. IEX assay

    1. Micropipettes (P2, P20, P200, P1000)

    2. NanoDrop spectrophotometer

    3. Thermomixer (Eppendorf, catalog number: 5382000015)

    4. Centrifuge (Eppendorf, catalog number: 5401000010)

    5. Äkta (Cytiva former GE Healthcare)

    6. Resource-Q or Mono-Q (Cytiva, catalog number: 17117701 or 17516601)

Software

  1. ImageQuantTL (optional) (GE Healthcarre)

  2. Typhoon FLA 7000 control software (GE Healthcare)

  3. PhotoShop CS6 (Adobe)

  4. Unicorn (Cytiva former GE Healthcare)

Procedure

  1. Diadenylate cyclase assay with radiolabeled ATP coupled with TLC

    1. Until Step A6, work on ice.

    2. Exchange the buffer of purified proteins to DisA cyclase buffer (e.g., via dialysis, see Notes).

    3. Determine the molar concentration of proteins (see Notes).

    4. Setup of the reaction:

      1. Calculate the volume of DisA required to achieve a concentration of 5 µM in 20 µl.

      2. Pipette the required volume of DisA into a 1.5 ml reaction tube (i.e., if the DisA stock has a concentration of 50 µM, you will need to take 2 µl).

      3. Fill up to a total volume of 19.5 µl with DisA cyclase buffer.

    5. From now on, work behind a radiation shield and use filter tips (see Notes).

    6. Add 0.5 µl [32P]-ATP to the reaction (final concentration: ~83 nM in 20 µl).

    7. Use 19.5 µl DisA cyclase buffer with 0.5 µl [32P]-ATP as a control for ATP migration.

    8. Incubate reactions for 60 min at 30 °C in the heat block.

    9. Pipette 5 µl 0.5 M EDTA, pH 8 into 1.5 ml reaction tubes (one tube per reaction).

    10. Add 5 µl of each reaction to a tube containing EDTA to stop diadenylate cyclase reaction.

    11. Spot the mixture from Step A10 on the cellulose side of a thin-layer (TL) plate (each spot should be ~1.5 cm from the bottom and lateral edges of the TL plate as well as other spots; also see Notes).

    12. Let the spots dry completely at room temperature (RT).

    13. Fill TLC chamber with TLC running buffer (buffer should be ~1 cm deep).

    14. Place the TL plate in the TLC chamber with spotted samples close to the buffer without touching it.

    15. Incubate TLC until the liquid front is ~1 cm from the upper edge of the TL plate (this can take up to ~2.5 h).

    16. Remove TL plate from the chamber and let dry at RT (overnight or at least 45 min in fume hood).

    17. Place an erased imaging plate (IP) into an exposure cassette.

    18. Wrap TL plate completely into plastic wrap (see Notes) and place with the cellulose side down on top of the IP for exposure.

    19. The exposure time may vary depending on the optimal signal-to-noise ratio, but ~30 min exposure will suffice in most cases.

    20. Remove the TL plate from the cassette and check cassette and IP for radioactive contamination using the contamination monitor (see Notes).

    21. If no contamination is present, scan the IP in the Typhoon FLA 7000 using the laser at 650 nm and IP filter.


  2. Phosphodiesterase assay with radiolabeled c-di-AMP coupled with TLC

    1. Until Step B7, work on ice.

    2. Exchange the buffer of purified AtaC to PDE reaction buffer (e.g., via dialysis, see Notes).

    3. Determine the molar concentration of AtaC (see Notes).

    4. If you want to perform competition with unlabeled nucleotides, proceed with Step B5, without competition proceed with Step B6.

    5. Setup of the reaction with competition:

      1. Calculate the volume of AtaC required to achieve a concentration of 100 nM in 20 µl.

      2. Pipette the required volume of AtaC into a 1.5 ml reaction tube (i.e., if the AtaC stock has a concentration of 1 µM, you will need to take 2 µl).

      3. Fill up to a total volume of 17.5 µl with PDE reaction buffer.

      4. Set up 1 mM solutions of each unlabeled nucleotide (c-di-AMP, c-di-GMP, cAMP) in ddH2O.

      5. Add 2 µl of each nucleotide per reaction in Step B5c.

      6. Set up a reaction with 2 µl ddH2O as negative control for competition.

      7. Proceed with Step B7.

    6. Setup of the reaction without competition:

      1. Calculate the volume of AtaC required to achieve a concentration of 100 nM in 20 µl.

      2. Pipette the required volume of AtaC into a 1.5 ml reaction tube (i.e., if the AtaC stock has a concentration of 1 µM, you will need to take 2 µl).

      3. Fill up to a total volume of 19.5 µl with PDE reaction buffer.

      4. Proceed with Step B7.

    7. From now on, work behind a radiation shield and use filter tips (see Notes).

    8. Dilute [32P]-c-di-AMP 1:20 in PDE reaction buffer and add 0.5 µl to the reaction (final concentration: ~2 nM in 20 µl).

    9. Use 19.5 µl PDE reaction buffer with 0.5 µl of 1:20 diluted [32P]-c-di-AMP as a control for c-di-AMP migration.

    10. Incubate reactions for 60 min at 30 °C in the heat block.

    11. To stop enzymatic reaction, incubate at 95 °C for 5 min.

    12. Centrifuge reactions at maximal speed for 3 min.

    13. Spot 5 µl supernatant of each reaction on the cellulose side of a thin-layer (TL) plate (each spot should be ~1.5 cm from the bottom and lateral edges of the TL plate and other spots).

    14. Let the spots dry completely at room temperature (RT).

    15. Fill TLC chamber with TLC running buffer (buffer should be ~1 cm high).

    16. Place the TL plate in the TLC chamber with spotted samples close to the buffer without touching it.

    17. Incubate TLC until the liquid front is ~1 cm from the upper edge of the TL plate (this can take up to ~2.5 h).

    18. Remove TL plate from the chamber and let dry at RT (overnight or at least 45 min in fume hood).

    19. Place an erased imaging plate (IP) into an exposure cassette.

    20. Wrap TL plate completely into plastic wrap (see Notes) and place with the cellulose side down on top of the IP for exposure.

    21. The exposure time may vary depending on the optimal signal-to-noise ratio, but ~60 min exposure will suffice in most cases.

    22. Remove the TL plate from the cassette and check cassette and IP for radioactive contamination using the contamination monitor.

    1. If no contamination is present, scan the IP in the Typhoon FLA 7000 using the laser at 650 nm and IP filter.


  3. IEX AtaC activity assay

    1. Set up 100 µl reactions containing 100 nM protein, 62.5 µM-2,000 µM substrate (e.g., c-di-AMP) and 1x reaction buffer. Control reactions containing protein or substrate only are necessary to exclude nonspecific results.

    2. Incubate the reaction at 37 °C for 1 h.

    3. Stop the reaction by transferring the complete reaction mix into a Microcon-30kDa Centrifugal Filter Unit and spinning for 10 min at 14,000 x g. This ultrafiltration step separates the protein from its reaction products, which are obtained in the filtrate.

    4. Repeat Step C3 if there is still liquid in the filter.

    5. Wash the filter by adding 100 µl running buffer A and repeating Step C3.

    6. Fill up the filtrate with running buffer A to 500 µl.

    7. Equilibrate an ion exchange column (1 ml Resource-Q or Mono-Q) in running buffer A.

    8. Inject the sample from step 6 and use a linear gradient from running buffer A to B (i.e., 0-40% B, 20 CV). Set the detection wavelength on 260 nm or 280 nm, if 260 nm is not possible.

Data analysis

  1. TLC assay

    Images scanned in the Typhoon FLA 7000 sometimes require adjustment of contrast. If necessary, open the scanned file in PhotoShop and modify spot intensity for the whole picture via “Image” → “Adjustments” → “Levels” until an optimal contrast is reached. Exemplary DAC and PDE assays separated by TLC are shown in Figure 2.



    Figure 2. Thin-layer chromatography (TLC) of a diadenylate cyclase (A) and a c-di-AMP phosphodiesterase assay (B). A. Diadenylate cyclase (DAC) assay with 5 µM of purified S. venezuelae wild-type DisA (DisASven, lanes 2 and 3) and inactive DisASven mutant variant (DisAD86A, lanes 4 and 5) incubated with 83 nM [32P]-ATP at 30 °C. After 30 and 60 min samples were taken and inactivated by mixing with equal volume of 0.5 M EDTA, pH 8. Inactivated reactions were spotted on a thin-layer (TL) plate and separated via TLC. After drying, the TL plate was exposed for 30 min to an imaging plate which was subsequently scanned using Typhoon FLA 7000. B. subtilis DisA (DisABsu, lane 6) was used as a positive control for DAC reaction. Lane 1 shows migration of [32P]-ATP in absence of enzymes. All samples were separated on the same TL plate and the picture was cropped to the relevant lanes. In contrast to the inactive DisAD86A, DisASven synthesizes [32P]-c-di-AMP out of [32P]-ATP as indicated by formation of a product that has the similar size like [32P]-c-di-AMP produced by DisABsu, which is a characterized DAC. B. Phosphodiesterase assay of S. venezuelae AtaC with competition with unlabeled nucleotides (lanes 3 to 5). 100 nM of purified protein was supplemented with 100 µM unlabeled c-di-AMP, c-di-GMP and cAMP (or ddH2O as control, lane 2) before incubation with 2 nM [32P]-c-di-AMP at 30 °C. After 60 min samples were taken and heat-inactivated. Precipitated protein was removed by centrifugation and supernatants of the inactivated reactions were spotted on a TL plate and separated via TLC. After drying, the TL plate was exposed for 60 min to an imaging plate which was subsequently scanned using Typhoon FLA 7000. Lane 1 shows migration of [32P]-c-di-AMP in absence of enzyme. AtaC cleaves [32P]-c-di-AMP to [32P]-AMP using [32P]-pApA as an intermediate cleavage product (lane 2). This reaction is specific since unlabeled c-di-AMP significantly inhibits, and thus outcompetes [32P]-c-di-AMP turnover (lane 3) whereas c-di-GMP and cAMP have no effect (lanes 4 and 5).


  2. IEX assay

    The IEX assay can also be used for quantification of the hydrolysis turnover for c-di-AMP. In this case, a peak integration of the respective nucleotide peaks has to be performed using the Äkta software Unicorn (Cytiva former GE Healthcare) or any other mathematical analysis software. From the ratio of the peak areas of substrate and product, a percentage of hydrolyzed substrate or formed product can be calculated. Figure 3 shows exemplary IEX separation of c-di-AMP, 5′-pApA and AMP standards. Figure 4 demonstrates the interfering peaks of c-di-AMP and 5′-pApA after IEX separation of a c-di-AMP PDE assay with AtaC and the removal of 5′-pApA from the reaction by TmPDE.



    Figure 3. Ion-Exchange Chromatography (IEX) standards for 100 µl solutions containing 200 µM c-di-AMP, 5′-pApA or AMP



    Figure 4. Ion-Exchange Chromatography (IEX) of a c-di-AMP phosphodiesterase assay. A. A 100 µl reaction containing 1 µM S. venezuelae AtaC + 400 µM c-di-AMP incubated for 1 h at 37 °C was stopped by ultrafiltration and separated via IEX. B. A similar reaction in a coupled assay with TmPDE to separate the peaks of c-di-AMP and 5′-pApA. A 100 µL reaction containing 1 µM S. venezuelae AtaC + 400 µM c-di-AMP incubated for 1 h at 37 °C was stopped by ultrafiltration, followed by the addition of 100 nM TmPDE which was stopped by ultrafiltration after incubation for 1 h at 37 °C and separated via IEX.

Notes

  1. General comments on TLC assays

    1. Buffer exchange is needed to replace the elution buffer in protein eluates to the proper reaction buffers (DisA cyclase buffer or PDE reaction buffer) and can be performed with many methods: e.g., size exclusion chromatography, ultrafiltration or dialysis. In our lab, we use dialysis, which however has no advantages or disadvantages compared to other methods for buffer exchange. You can choose a method that suits you best.

    2. The exact method to determine the protein concentration is not crucial to perform the TLC assays and can be achieved by many methods: e.g., photometric measurement at 280 nm, Bradford assay or 1D SDS gel densitometry. In our lab, we routinely use the latter, which however has no advantages or disadvantages compared to other methods. Feel free to use any method that is convenient for you.

    3. Enzymatic assays with radiolabeled nucleotides should be performed only by trained individuals with sufficient protection equipment.

    4. Usage of filter tips is strongly recommended to avoid contamination of micropipettes with radioactive material.

    5. The given enzyme concentrations were optimized for purified DisA (S. venezuelae and B. subtilis) and purified AtaC (S. venezuelae). Enzymes from other bacteria may require different concentrations. If you work with a new protein, first test enzymatic activity with 1 to 5 µM purified protein and subsequently determine the optimal protein concentrations by titration.

    6. Procedure section, Step A11: spotting the whole 10 µl mixture at once may result in blending of the single spots before drying. To avoid that, you can spot 3 µl + 3 µl + 4 µl with drying of spots between each volume spotted.

    7. The TL plate can be stored at RT for at least 3 days without significant loss of signal quality and can be used multiple times for exposure to erased imaging plates. If you are working with substrates with lower radioactive activity than indicated in the materials section, it might be necessary to prolong the exposure times of the imaging plate.

    8. The use of plastic wrap for the TL plate reduces the risk of radioactive contamination of the imaging plate and cassette. You may place an additional layer of plastic wrap on the imaging plate to further reduce the risk.

    9. Imaging plates can be erased by exposure to a light source (e.g., for 10 min using the image eraser, Molecular Dynamics, model: 410A) and reused. Consequently, after removing the TL plate from the cassette, avoid long exposure of the imaging plate to ambient light prior to scanning.


  2. General comments on IEX assay

    1. c-di-AMP forms long non-symmetrical Peaks on IEX columns and can therefore overlay with other Peaks (see also comment 3). Thus, if a RP-HPLC system is available, it is the preferred method to use instead.

    2. Basic knowledge of Äkta systems is required.

    3. If the c-di-AMP conversion has to be quantified by peak integration, the overlay of the peaks from 5′-pApA and c-di-AMP has to be considered. In this case, a second enzyme, which can only degrade 5′-pApA but not c-di-AMP (e.g., TmPDE [Drexler et al., 2017]), has to be added after ultrafiltration. Add TmPDE to a final concentration of 100 nM and incubate at 37 °C for 1 h. Repeat the procedure section paragraph C starting from step 3.

    4. The precise elution point of the nucleotides on an IEX column is very sensitive to salt, pH, temperature and also the Äkta system. For optimal comparison, assays should be performed with the same buffer and on the same Äkta system.

Recipes

  1. DisA cyclase reaction buffer, modified from Christen et al. (2005)

    25 mM Tris-HCl pH = 8

    250 mM NaCl

    10 mM MgCl2

    5 mM β-mercaptoethanol

    10% glycerol

  2. PDE reaction buffer, modified from Huynh et al. (2015)

    20 mM Tris-HCl pH = 7.5

    50 mM NaCl

    1 mM MnCl2

  3. TLC running buffer, modified from Christen et al. (2005)

    Saturated (NH4)2SO4 and 1.5 M KH2PO4 pH = 3.6 (mixed in a ratio of 1:1.5 v/v)

  4. 10x AtaC reaction buffer

    500 mM Tris pH = 7.5

    1,000 mM NaCl

    1 mM MnCl2

  5. IEX running buffer A

    50 mM Tris pH = 9

  6. IEX running buffer B

    50 mM Tris pH = 9

    1,000 mM NaCl

Acknowledgments

Research in the Witte lab is funded by DFG Grant GRK1721 and the DFG Priority Program SPP 1879 (Grant WI 3717/3-1). Research in the Tschowri lab is funded by the DFG Emmy Noether Program (Grant TS 325/1-1) and the DFG Priority Program SPP 1879 (Grants TS 325/2-1 and TS 325/2-2).

Competing interests

The authors declare no competing interests.

References

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简介

[摘要]所有活细胞均使用环状核苷酸作为第二信使,以进行信号传感和转导。环状二-3 ' ,5 ' -腺苷一磷酸(C-二-AMP)主要涉及细菌和控制euryarcheal osmoadaptation并且由产生diadenylate环化酶从ATP的两个分子。特定的磷酸二酯酶将c-di-AMP水解为线性磷酸腺苷腺苷5'-pApA或AMP。可以使用包括高效液相色谱(HPLC),薄层色谱(TLC)和离子交换色谱(IEX)在内的各种方法来确定c-di-AMP合成和降解酶的活性。在这里,我们详细描述了TLC和IEX方法适合于表征diadenylate环化酶DISA和磷酸二酯酶ATAC从链霉菌venezuelae 。TLC可以快速,轻松地分离放射性标记的底物和产品,而IEX避免了潜在危险的放射性底物的利用,如果没有HPLC系统,则可以用作良好的替代品。与TLC分析不同,无法使用IEX分析并行分析样品,因此更加耗时。


[背景]环核苷酸第二信使是原核和真核信号通路中的关键分子。环状二-3 ' ,5 ' -腺苷一磷酸(三二- AMP)是一种细菌第二信使与许多重要的功能,如渗压剂动态平衡调节,细胞壁代谢,生物膜的形成,DNA完整,孢子形成,毒力和生长(Fahmi et al。,2017)。这是特别令人感兴趣的,因为它存在于许多人类病原体,如金黄色葡萄球菌,结核分枝杆菌和肺炎链球菌和被发现是正常生长在大多数物种必要条件(伍德沃德等人,2010;里根等人, 2011; Luo和Helmann ,2012; Mehne等,2013; Whiteley等,2015)。有趣的是,还积累的C-二AMP导致生长缺陷(白等人,2013; Mehne等人,2013; Latoscha 。等人,2020)。因此,这个第二信使的水平必须进行严格的监管。c-di-AMP由二腺苷酸环化酶(DAC)的两个ATP分子合成,并被特定的磷酸二酯酶(PDE)降解为5'-pApA和/或两个AMP分子(图1)(Fahmi等,2017; (Yin等,2020)。



图1.合成和在C-二AMP降解S. venezuelae 。二腺苷酸环化酶DisA使用镁离子(Mg 2+ )作为辅因子,由两个ATP分子合成c-di-AMP,释放出作为副产物的两个焦磷酸盐(PP i )(Witte等,2008)。c-di-AMP被磷酸二酯酶AtaC降解为5'-pApA和两个AMP分子,这需要锰离子(Mn 2+ )作为辅助因子(Latoscha et al 。,2020 )。



通过DAC结构域合成c-di-AMP被鉴定为五种不同的蛋白质类型,称为DisA ,CdaA ,CdaS ,CdaM和CdaZ 。大多数细菌都含有用于c-di-AMP合成的DisA或CdaA ,而一些仅属于芽孢杆菌属的物种并行拥有DisA ,CdaA和CdaS (Commichau等人,2019)。这些蛋白质的酶活性可能受不同条件的影响。最普遍的DAC CdaA由胞外调节剂CdaR和磷酸葡糖胺变位酶GlmM调节(Tosi等,2019; Gibhardt等,2020)。与此相反,DISA合成的c-二-AMP组成除非它结合支化或受损的DNA(维特等人,2008),其结果在孢子形成延迟(Bejerano-Sagie等人,2006; Oppenheimer- Shaanan 。等人, 2011)。因此,c-di-AMP的合成可能受到许多不同的环境条件的影响,这些条件尚待详细了解。对于c-di-AMP降解,到目前为止已鉴定出两大类特定的PDE,称为DHH型和HD型PDE,它们根据保守的催化氨基酸基序Asp-His-His和His-命名。在其各自的活动站点中的asp。DHH型PDE进一步分为多域膜偶联蛋白,如GdpP (Pde1)和DhhP型可溶性独立催化域蛋白(Pde2)(Rao等,2010; Bai等,2013; Ye等。,2014)。HD型PDE是7TMR-HD家族(具有HD结构域的7个跨膜结构域受体)的膜偶联成员,称为P gpH (Gundlach等,2015; Huynh等,2015)。既GDPP和PGPH专门水解C-二AMP为5'- PAPA,其通过进一步降解成AMP DhhP或纳米RNA酶,如NrnA 。DhhP PDEs通常在一个物种中与GdpP或PgpH平行出现,并被证明是造成5'-pApA到AMP的第二个降解步骤的原因(Bowman等人,2016; Drexler等人,2017; Konno等人。,2018)。相比之下,某些物种,例如结核分枝杆菌和耻垢分枝杆菌仅含有DhhP同源物,它们被证明能水解c-di-AMP和5'- pApA(Tang等,2015; He等,2016)。 。然而,来自放线菌门的许多物种,包括链霉菌,都不具有任何主要的c-di-AMP特异性PDE类(Corrigan和Gründling ,2013; Yin等人,2020)。相反,委内瑞斯链霉菌利用另一种PDE降解c-di-AMP。在我们最近的研究中,我们采用了不同的生化方法来表征DisA (放线菌中编码的唯一DAC域蛋白)以及AtaC (放线菌PDE靶向c-di-AMP),它是c-di-AMP新家族的创始成员。 di-AMP特有的PDE,主要存在于放线菌中(Latoscha et al。,2020)。

为了准确表征此信号通路中的蛋白质,需要鲁棒且灵敏的底物特异性检测方法。在本协议中,我们描述了两种用于c-di-AMP合成或降解分析的优化方法:具有放射性标记核苷酸的酶法测定的薄层色谱法(TLC)和离子交换色谱法(IEX)。

的TLC分析建立最初研究的活动柄杆菌新月二鸟苷酸环化酶(DGC)PLED PDE和c二GMP特异性CC3396(保罗等人;克里森,2004等为主,2005)上的方法,发表罗斯等。(1987)。DGC和c-di-GMP特异性PDE放射性检测结合TLC的详细方案最近已发布(Kazmierczak ,2017a和2017b)。在过去的几年,随着加上TLC放射性标记核苷酸的酶测定法已成功地用于从革兰氏阴性和革兰氏阳性的c-二GMP特异性的酶的表征细菌(Kazmierczak等人,2006;德雷奥。等人, 2011;林登贝格等人,2013;铝巴萨姆。等人,2018)以及在DAC的DISA从枯草芽孢杆菌和栖热袍(维特等人,2008; Torres的。等人,2019)。与高效液相色谱(HPLC)等其他方法相比,TLC分析可同时从多个反应中分离底物和产物。此外,如果在反应中添加冷核苷酸作为酶活性的竞争者,则可以得出有关底物特异性的直接结论(Paul等,2004; Christen等,2005;Lindenberg等,2013)。如果需要,可以对放射性产物的形成进行定量,因为在TLC研发后仅可视化了放射性标记的核苷酸,而未可视化的竞争剂(Ross等,1987; Paul等,2004; Christen等, 2005年)。在我们最近的研究中,我们使用的TLC分析来分析由两个合成的c-二-AMP的S. venezuelae DISA通过和特异性的c-二AMP降解的ATAC (Latoscha等人,2020)。

IEX被用作进一步表征AtaC活性的独立方法(Latoscha等,2020)。在该测定中,带负电荷的分子(例如核苷酸)与带正电荷的色谱柱材料结合,并可以通过盐梯度洗脱。取决于它们与色谱柱的结合亲和力,分子可以以不同的盐浓度洗脱并可以分离。如果没有其他用于小分子的色谱系统,例如反相HPLC(RP-HPLC),则该测定法尤为重要。Li等人首先通过离子交换色谱法分离了环状二核苷酸及其相关的反应中间体5'-pNpN或NMP 。(201 3)分析环状GMP-AMP合酶(cGAS )的反应产物。该方法被Luecke等人进一步使用。Holleufer和Hartmann(2018)(2017)详细描述了各自的检测方法。该方法适于在降解PDE的特征的c-二-AMP的途径(德雷克斯勒等人,2017),并稍作修改用于表征本测定ATAC (Latoscha ê吨人,2020) 。原则上,该IEX分析可用于分析大多数环状二核苷酸合成或降解酶的活性。但是,RP-HPLC系统可以更快,更精确地分离样品,如果有的话,则RP-HPLC将是首选的色谱方法,如一些报告中所述(Oppenheimer- Shaanan等人,2011年)。 ; Bai等人,2012; Witte等人,2013; Huynh等人,2015; Tang等人,2015)。

关键字:环二腺苷酸, 二腺苷酸环化酶, DAC, DisA, 磷酸二酯酶, PDE, AtaC, 酶法测定

材料和试剂
缓冲区交换的材料(请参阅注释)
45 mm的固定夹(Carl Roth,货号:H277.1)
透析膜Spectra / Por 7 MWCO 10,000,45毫米(Carl Roth,目录号:E872.1)
玻璃吸管:5,10,20和25毫升(大号abdirect ,产品目录号:021.01.005,355.050.110,355.050.120,355.050.125)
移液器吸头(Sarstedt ,目录号:70.760.012、70.762、70.3020和Biozym ,目录号:VZ0001X)
1.5 ml反应管(Sarstedt ,目录号:72.690.001)
双蒸馏水(ddH 2 O)(Kerndl ,目录号:22501)
六水合氯化镁(MgCl 2 )(卡尔·罗斯,目录号:2189.1)
四水合氯化锰(MnCl 2 )(Carl Roth,目录号:T881.1)
氯化钠(NaCl)(VWR,目录号:27810.295DB)
Tris-base(Carl Roth,货号:4855.2)
甘油(Carl Roth,目录号:3783.2)
β-巯基乙醇(Sigma-Aldrich,目录号:M6250)

用于蛋白质浓度测定的材料(请参阅注释)
1.5 ml反应管(Sarstedt ,目录号:72.690.001)
间隔板和短板(Bio-Rad,目录号:1653310和1653308)
移液器吸头(Sarstedt ,目录号:70.760.012、70.762、70.3020和Biozym ,目录号:VZ0001X)
Tris-base(Carl Roth,货号:4855.2)
甘氨酸(卡尔·罗斯,目录号:0079.4)
有色预染色的标记物(NEB,目录号:P7719S)
LMW标记(GE Healthcare,目录号:17-0446-01)
双蒸馏水(ddH 2 O)(Kerndl ,目录号:22501)
Rotiphorese (Carl Roth,目录号:3029.1)
过二硫酸铵(Carl Roth,目录号:9178.1)
TEMED(卡尔·罗斯,目录号:2367.3)
甘油(Carl Roth,目录号:3783.2)
月桂基硫酸钠(Carl Roth,目录号:4360.2)
溴酚蓝(Carl Roth,目录号:A512.1)
β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
2-丙醇(Carl Roth,目录号:6752.5)
醋酸(Carl Roth,目录号:6755.1)
考马斯蓝R 250(Carl Roth,货号:3862.2)

带有放射性标记核苷酸的酶促测定的薄层色谱法(TLC)
移液器吸头(Sarstedt ,目录号:70.760.012和Biozym ,目录号:VZ0001X)
移液器过滤嘴(Sarstedt ,目录号:70.1116.210)
1.5 ml反应管(Sarstedt ,目录号:72.690.001)
薄板(Macherey -Nagel,POLYGRAM CEL 300 PEI,目录号801053)
荧光成像板(Fujifilm,BAS-IP SR 2025)
保鲜膜(从杂货店获得的任何类型)
曝光盒(GE Healthcare,目录号:63-0035-44)
纯化的DISA从S. venezuelae或枯草芽孢杆菌(维特等人,2008; Latoscha 。等人,2020)
纯化非活动DISA变体(DISA D86A )从S. venezuelae (Latoscha等人,2020)
纯化ATAC从S. venezuelae (Latoscha等人,2020)
9.25 MBq [α- 32 P] -ATP(Hartmann Analytik ,目录号:FP-207)
9.25活度[ 32 P] -c二AMP(哈特曼Analytik的,目录号:FP-C517;一个纯化的c-二AMP生成酶,例如,枯草芽孢杆菌DISA ,必须提供给公司为[ 32 P ] -c-di-AMP根据要求合成)
对于磷酸二酯酶竞争测定:
c-di-AMP(BioLog ,目录号:C 088)
c-di-GMP(BioLog ,目录号:C 057)
cAMP(Sigma-Aldrich,目录号:A6885)
六水合氯化镁(MgCl 2 )(卡尔·罗斯,目录号:2189.1)
四水合氯化锰(MnCl 2 )(Carl Roth,目录号:T881.1)
氯化钠(NaCl)(VWR,目录号:27810.295DB)
Tris-base(Carl Roth,货号:4855.2)
盐酸(Carl Roth,目录号:4625.2)
甘油(Carl Roth,目录号:3783.2)
β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
双蒸馏水(ddH 2 O)(Kerndl ,目录号:22501)
0.5 M EDTA,pH 8(PanReac AppliChem,目录号:A4892)
硫酸铵((NH 4 )2 SO 4 )(VWR,目录号:21333.296DB)
磷酸一氢钾(KH 2 PO 4 )(卡尔·罗斯,目录号:P018.2)
DisA环化酶缓冲液(请参见食谱)
PDE反应缓冲液(请参阅配方)
TLC运行缓冲区(请参阅配方)

IEX活性测定
移液器吸头(Sarstedt ,目录号:70 .1130.600和70.760.502)
1.5 ml反应管(Sarstedt ,目录号:72.690.001)
Microcon-30kDa离心过滤器(Merck Millipore,目录号:MRCF0R030)
纯化ATAC从S. venezuelae (Latoscha等人,2020)
纯化TMPDE从T. maritima的(德雷克斯勒等人,2017)
c-di-AMP(BioLog ,目录号:C 088)
四水合氯化锰(MnCl 2 )(Carl Roth,目录号:T881.3)
氯化钠(Merck Millipore,目录号:1.06404.5000)
Tris-base(Carl Roth,货号:4855.2)
25%盐酸(VWR,目录号:20257.296)
10x AtaC反应缓冲液(请参阅食谱)
IEX运行缓冲区A(请参阅配方)
IEX运行缓冲区B(请参见配方)

设备
缓冲区交换设备(请参阅注释)
5,000毫升量筒(Carl Roth,目录号:0780.1)
电磁棒(Carl Roth,目录号:C267.1)
移液辅助宏(Carl Roth,目录号:X478.1)
微量移液器(P20,P200,P1000)
磁力搅拌器(Heidolph ,型号:MR 2000)
离心机(Eppendorf,型号:5427 R,货号:5409000010)
4°C室

您选择的蛋白质浓度测定设备(请参阅注释)
Mini-PROTEAN Tetra Cell(Bio-Rad,货号:1658000EDU)
加热块(Labnet International,型号:AccuBlock TM数字干浴锅,目录号:D1302-230V)
离心机(Eppendorf,型号:5427 R,货号:5409000010)
电源(Bio-Rad,目录号:1645050)
带有CCD摄像机的成像仪(GE Healthcare,型号:ImageQuant LAS 4000)
微量移液器(P2,P10,P20)

放射性标记核苷酸酶促测定的薄层色谱
1.微量移液器(P2,P10,P20)     
2.加热块(Labnet International,型号:AccuBlock TM数字干浴锅,目录号:D1302-230V)     
3.离心机(Eppendorf,型号:5427 R,目录号:5409000010)     
4.污染监测仪(Berthold,型号:LB 122)     
5.生物分子成像仪(GE Healthcare,型号:Typhoon FLA 7000,目录号:28-9558-09)     
6.图像擦除器(分子动力学,型号:410A)     
7.丙烯酸台式Beta辐射防护屏(ThermoFisher Scientific,目录号:6700-2418)     
8. TLC室(Fisher Scientific,目录号:06-815-187)     
9.通风柜     

IEX测定
微量移液器(P2,P20,P200,P1000)
NanoDrop分光光度计
Thermomixer(Eppendorf,目录号:5382000015)
离心机(埃彭多夫(Eppendorf),目录号:5401000010)
Äkta (Cytiva,前GE Healthcare)
Resource-Q或Mono-Q(Cytiva ,目录号:17117701或17516601)

软件



ImageQuantTL (可选)(GE Healthcarre )
Typhoon FLA 7000控制软件(GE Healthcare)
PhotoShop CS6(Adobe)
独角兽(Cytiva,前GE Healthcare)

程序
放射性标记的ATP与TLC联用的二腺苷酸环化酶测定
直到步骤A6,在冰上工作。
将纯化蛋白的缓冲液交换为DisA环化酶缓冲液(例如,通过透析,请参见“注释”)。
确定蛋白质的摩尔浓度(请参阅注释)。
反应的建立:             
计算在20 µl中达到5 µM浓度所需的DisA体积。
将所需量的DisA移液到1.5 ml反应管中(即,如果DisA储备液的浓度为50 µM,则需要取2 µl)。
用DisA环化酶缓冲液填充总体积为19.5 µl 。
从现在开始,在防辐射罩后面工作并使用过滤嘴(请参阅“注释”)。
向反应中添加0.5 µl [ 32 P] -ATP(最终浓度:20 µl中约83 nM )。
使用19.5 µl DisA环化酶缓冲液和0.5 µl [ 32 P] -ATP作为ATP迁移的对照。
在加热块中于30°C孵育反应60分钟。
吸取5 µl 0.5 M EDTA,pH 8到1.5 ml反应管中(每个反应一个管)。
将每个反应5微升添加到装有EDTA的试管中,以终止二腺苷酸环化酶反应。
将步骤A10的混合物点在薄板(TL)板的纤维素侧上(每个点应距TL板的底部和侧面边缘以及其他斑点约1.5 cm;请参见注释)。
让斑点在室温(RT)下完全干燥。
用TLC运行缓冲液(缓冲液应约1厘米深)填充TLC腔室。
将TL板放置在TLC室中,使有斑点的样品靠近缓冲液,而不接触缓冲液。
孵育TLC,直到液体前沿距TL板的上边缘约1 cm(这可能需要约2.5 h)。
从室中取出TL板,并在室温下干燥(过夜或在通风橱中至少45分钟)。
将已擦除的成像板(IP)放入曝光盒中。
将TL板完全包裹在保鲜膜中(请参见注释),并将纤维素面朝下放在IP顶部以进行曝光。
所述ë xposure时间可以根据最佳信噪比而变化,但约30分钟的曝光将在大多数情况下是足够的。
从暗盒中取出TL板,并使用污染监测仪检查暗盒和IP是否存在放射性污染(请参阅“注释”)。
如果没有污染,请使用650 nm激光和IP过滤器扫描Typhoon FLA 7000中的IP。

放射性标记的c-di-AMP与TLC联用测定磷酸二酯酶
直到步骤B7,在冰上工作。
将纯化的AtaC缓冲液交换为PDE反应缓冲液(例如,通过透析,请参见注释)。
确定AtaC的摩尔浓度(请参见注释)。
如果要与未标记的核苷酸竞争,请继续执行步骤B5,而没有竞争则继续进行步骤B6。
建立竞争反应:             
计算在20 µl中达到100 nM浓度所需的AtaC体积。
将所需体积的AtaC移液到1.5 ml反应管中(即,如果AtaC储备液的浓度为1 µM,则需要取2 µl)。             
用PDE反应缓冲液填充总体积为17.5 µl。             
在ddH 2 O中设置每个未标记核苷酸(c-di-AMP,c-di-GMP,cAMP)的1 mM溶液。
在步骤B5c中,每个反应添加2 µl每个核苷酸。              
用2 µl ddH 2 O设置反应作为竞争的阴性对照。
继续执行步骤B7。
无需竞争即可建立反应:
计算在20 µl中达到100 nM浓度所需的AtaC体积。             
将所需体积的AtaC移液到1.5 ml反应管中(即,如果AtaC储备液的浓度为1 µM,则需要取2 µl)。
用PDE反应缓冲液填充总体积为19.5 µl。
继续执行步骤B7。
从现在开始,在防辐射罩后面工作并使用过滤嘴(请参阅“注释”)。
在PDE反应缓冲液中稀释[ 32 P] -c-di-AMP 1:20并向反应中添加0.5 µl(最终浓度:20 µl中约2 nM )。
使用19.5 µl PDE反应缓冲液和0.5 µl 1:20稀释的[ 32 P] -c-di-AMP稀释液作为c-di-AMP迁移的对照。
在加热块中于30°C孵育反应60分钟。
要停止酶促反应,请在95°C下孵育5分钟。
以最大速度离心反应3分钟。
在薄层(TL)板的纤维素侧上点每个反应的5 µl上清液(每个点应距TL板的底部和侧面边缘及其他点约1.5 cm)。
让斑点在室温(RT)下完全干燥。
用TLC运行缓冲液(缓冲液应高约1厘米)填充TLC腔室。
将TL板放置在TLC室中,使有斑点的样品靠近缓冲液,而不接触缓冲液。
孵育TLC,直到液体前沿距TL板的上边缘约1 cm(这可能需要约2.5 h)。
从室中取出TL板,并在室温下干燥(过夜或在通风橱中至少45分钟)。
将已擦除的成像板(IP)放入曝光盒中。
将TL板完全包裹在保鲜膜中(请参见注释),并将纤维素面朝下放在IP顶部以进行曝光。
电子xposure时间可以根据最佳信噪比而变化,但〜60分钟的曝光将在大多数情况下是足够的。
从暗盒中取出TL板,并使用污染监测仪检查暗盒和IP是否存在放射性污染。
如果没有污染,请使用650 nm激光和IP过滤器扫描Typhoon FLA 7000中的IP。

IEX AtaC活性测定
设置100 µl反应,其中包含100 nM蛋白,62.5 µM-2,000 µM底物(例如c-di-AMP)和1x反应缓冲液。仅包含蛋白质或底物的对照反应是排除非特异性结果所必需的。
将反应液在37°C下孵育1小时。
通过将完整的反应混合物转移到Microcon-30kDa离心过滤器中并以14,000 xg旋转10分钟来终止反应。该超滤步骤将蛋白质与其在滤液中获得的反应产物分离。
如果过滤器中仍然有液体,请重复步骤C3 。
加入100 µl运行缓冲液A并重复步骤C3,以洗涤过滤器。
用运行缓冲液A充满滤液至500 µl。
在运行缓冲液A中平衡离子交换柱(1 ml Resource-Q或Mono-Q)。
从步骤6注入样品,并使用从运行缓冲液A到B的线性梯度(即0-40%B,20 CV)。如果无法使用260 nm,则将检测波长设置为260 nm或280 nm。

数据分析



薄层色谱法
在台风FLA 7000中扫描的图像有时需要调整对比度。如有必要,请在PhotoShop中打开扫描的文件,然后通过“图像”→“调整”→“色阶”修改整个图片的斑点强度,直至达到最佳对比度。由TLC分离的示例性DAC和PDE分析如图2所示。           





图2.二腺苷酸环化酶(A)的薄层色谱(TLC)和c-di-AMP磷酸二酯酶测定(B)。A. Diadenylate环化酶(DAC)测定与提纯的5μM S. venezuelae野生型DISA (DISA斯文,泳道2和3)和非活动DISA斯文突变体变体(DISA D86A 83温育,泳道4和5)nM的[ 32 P] -ATP在30°C。后30个分钟和60分钟取样并通过灭活用等体积的0.5M EDTA混合,pH为8的灭活反应物点样于薄层(TL)和板通过TLC分离。干燥后,将TL板暴露于成像板上30分钟,然后使用Typhoon FLA 7000对其进行扫描。枯草芽孢杆菌DisA (DisA Bsu ,泳道6)用作DAC反应的阳性对照。泳道1显示[ 32 P] -ATP在没有酶的情况下的迁移。将所有样品在相同的TL板上分离,并将图片裁剪到相关通道。与无活性的DisA D86A相比,DisA Sven从[ 32 P] -ATP中合成了[ 32 P] -c-di-AMP,这是通过形成具有[ 32 P] -c-di相似大小的产物表明的-AMP产生由DISA BSU ,这是一个特征DAC。的磷酸二酯酶B.测定S. venezuelae ATAC与未标记的核苷酸(泳道3至5)的竞争。100 nM的纯化的蛋白质的瓦特为补充有100μM的未标记的c-二-AMP,C二-GMP和cAMP(或的DDH 2与2孵育前O作为控制,泳道2)nM的[ 32 P] -c二AMP在30°C下。60分钟后,取样并加热灭活。通过离心去除沉淀的蛋白质,并将灭活反应的上清液点在TL板上,并通过TLC分离。干燥后,将TL板暴露在成像板上60分钟,然后使用Typhoon FLA 7000对其进行扫描。泳道1显示[ 32 P] -c-di-AMP在没有酶的情况下的迁移。AtaC使用[ 32 P] -pApA作为中间裂解产物,将[ 32 P] -c-di-AMP裂解为[ 32 P] -AMP (泳道2)。该反应是特异性的,因为未标记的c-di-AMP显着抑制,因此胜过[ 32 P] -c-di-AMP转换(泳道3),而c-di-GMP和cAMP没有作用(泳道4和5)。



IEX测定
IEX测定也可以用于定量c-di-AMP的水解转换。在这种情况下,相应的核苷酸峰的峰积分必须使用进行ÄKTA软件麒麟(Cytiva前GE医疗集团)或任何其他数学分析软件。从底物和产物的峰面积之比,可以计算水解的底物或形成的产物的百分比。图3显示了c-di-AMP,5'-pApA和AMP标准品的示例IEX分离。图4显示的c-二-AMP和5'-PAPA的c二AMP PDE测定法使用的IEX分离后的干扰峰ATAC和从由反应中除去5'- PAPA的TMPDE 。







图3.包含200 µM c-di-AMP,5'-pApA或AMP的100 µl溶液的离子交换色谱(IEX)标准







图4. c-di-AMP磷酸二酯酶测定的离子交换色谱(IEX)。含有1μMA.将100μl反应S. venezuelae ATAC + 400μMC-二AMP温育1个小时,在37℃通过超滤停止并通过IEX分离。B.在与TmPDE的偶联测定中的相似反应,以分离c-di-AMP和5'-pApA的峰。含有1μMA 100μL反应S. venezuelae ATAC + 4 00μMC-二AMP温育1个小时,在37℃下通过超滤停止,随后b y中的另外100 nM的TMPDE将其温育之后停止,通过超滤进行在37°C下放置1 h,然后通过IEX分离。

笔记
关于TLC分析的一般评论
需要进行缓冲液交换,以将蛋白质洗脱液中的洗脱缓冲液替换为适当的反应缓冲液(DisA环化酶缓冲液或PDE反应缓冲液),并且可以通过多种方法进行:例如尺寸排阻色谱法,超滤或透析。在我们的实验室中,我们使用透析,但是与其他缓冲液交换方法相比,它没有优点或缺点。您可以选择最适合您的方法。
确定蛋白浓度的确切方法对于进行TLC测定并不重要,可以通过许多方法实现:例如,在280 nm处进行光度测量,Bradford测定或1D SDS凝胶密度测定。在我们的实验室中,我们通常使用后者,但是与其他方法相比,它没有优点或缺点。随意使用任何方便的方法。
具有放射性标记核苷酸的酶促测定只能由训练有素的具有足够防护设备的人员进行。
强烈建议使用过滤嘴,以避免微量移液器被放射性物质污染。
给定的酶浓度针对纯化的DisA (S。委内瑞拉和枯草芽孢杆菌)和纯化的AtaC (S。委内瑞拉)进行了优化。来自其他细菌的酶可能需要不同的浓度。如果您使用的是新蛋白质,请先测试1至5 µM纯化蛋白质的酶促活性,然后通过滴定确定最佳蛋白质浓度。
步骤A11的程序部分:一次点样整个10 µl混合物可能会导致干燥之前混合单个点。为避免这种情况,您可以点样3 µl + 3 µl + 4 µl,并在每个点样体积之间干燥点样。
TL板可以在室温下保存至少3天,而不会显着降低信号质量,并且可以多次用于曝光的已擦除成像板。如果您正在处理放射性低于材料部分中规定的底物,则可能需要延长成像板的曝光时间。
TL板使用保鲜膜可降低成像板和暗盒受到放射性污染的风险。您可以在成像板上放置一层保鲜膜,以进一步降低风险。
成像板可以通过暴露在光源下进行擦除(例如,使用图像擦除器,Molecular Dynamics,型号:410A持续10分钟),然后重复使用。因此,从纸盒中取出TL板后,请避免在扫描前将成像板长时间暴露在环境光下。

关于IEX分析的一般评论
c-di-AMP在IEX色谱柱上形成长的非对称峰,因此可以与其他峰重叠(另请参见注释3)。因此,如果有RP-HPLC系统,则首选使用该方法。
需要Äkta系统的基础知识。
如果C二-AMP的转换必须由峰积分,峰从5'爸爸覆盖和c-二AMP具有量化加以考虑。在这种情况下,第二酶,它只能劣化5'-PAPA但不是C二叔AMP(例如,TMPDE [德雷克斯勒等人,2017]),具有超滤之后加入。加入TmPDE至终浓度为100 nM,并在37°C下孵育1 h。从第3步开始重复过程C节。
上的IEX柱核苷酸的精确洗脱点是盐,PH值,温度非常敏感,并且还与AKTA系统。为了进行最佳比较,应使用相同的缓冲液和相同的Äkta系统进行测定。

菜谱

DisA环化酶反应缓冲液,由Christen等人修改。(2005年)
25 mM Tris-HCl pH = 8

250毫米氯化钠

10毫米MgCl 2

5 mMβ-巯基乙醇

10%甘油

PDE反应缓冲液,由Huynh等人修改。(2015年)
20 mM Tris-HCl pH = 7.5

50毫米氯化钠


1毫米MnCl 2

TLC运行缓冲区,由Christen等人修改。(2005年)
饱和(NH 4 )2 SO 4和1.5 M KH 2 PO 4 pH = 3.6(以1:1.5 v / v的比例混合)

10x AtaC反应缓冲液
500 mM Tris pH = 7.5
1,000毫米氯化钠
1毫米MnCl 2
IEX运行缓冲区A
50 mM Tris pH = 9

IEX运行缓冲区B
50 mM Tris pH = 9
1,000毫米氯化钠

致谢
Witte实验室的研究由DFG Grant GRK1721和DFG Priority Program SPP 1879(格兰特WI 3717 / 3-1)资助。研究在Tschowri实验室由DFG资助艾美奖诺特计划(批准TS 325 / 1-1)和DFG优先项目计划1879年SPP(拨款TS 325 / 2-1和TS 325 / 2-2)

利益争夺
作者宣称没有利益冲突。

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引用:Latoscha, A., Drexler, D. J., Witte, G. and Tschowri, N. (2021). Assessment of Diadenylate Cyclase and c-di-AMP-phosphodiesterase Activities Using Thin-layer and Ion Exchange Chromatography. Bio-protocol 11(1): e3870. DOI: 10.21769/BioProtoc.3870.
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