Measuring CD38 Hydrolase and Cyclase Activities: 1,N⁶-Ethenonicotinamide Adenine Dinucleotide (ε-NAD) and Nicotinamide Guanine Dinucleotide (NGD) Fluorescence-based Methods

Materials and Reagents

1. Plastic tips 1,000 (Thermo Scientific^®, Molecular Bioproducts, catalog number: 3101 )
   
2. Plastic tips 200 (Thermo Fisher Scientific, Molecular Bioproducts, catalog number: 3551 )
   
3. 96-well plate (Microfluor 1 White flat-bottom plate) (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 7705 )
   
4. 1.5 ml tubes
   
5. 60 mm culture dishes (Fisher Scientific, Fisherbrand^TM, catalog number: FB012921 )
   
6. Tissues of interest: any tissue can be used to measure NAD⁺/NADH levels
   
7. Cells of interest: we usually use A549, JURKAT, Patu 9888T to measure NAD⁺/NADH levels
   
8. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
   
9. Sucrose (Sigma-Aldrich, catalog number: S0389-1KG )
   
10. Tris Base (Trizma^® base, Sigma-Aldrich, catalog number: T6066 )
    
11. Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
    
12. CD38 human recombinant enzyme (R&D Systems, catalog number: 2404-AC-010 )
    
13. CD38 inhibitor (Merck, Calbiochem, catalog number: 538763 )
    
14. Anti-CD38 antibody–Isatuximab (Creative-Biolabs, catalog number: TAB-432CQ )
    
15. Nicotinamide guanine dinucleotide sodium salt (NGD) (Sigma-Aldrich, catalog number: N5131 )
    
16. Nicotinamide 1, N6-ethenoadenine dinucleotide (ε-NAD) (Santa Cruz Biotechnology, catalog number: sc-215559 )
    
17. MES (Sigma-Aldrich, catalog number: M3671 )
    
18. Sodium chloride (Sigma-Aldrich, catalog number: S7653 )
    
19. Nanopure water
    
20. HCl
    
21. Sucrose Buffer (see Recipes)
    
22. rhCD38 enzyme buffer (see Recipes)
    
    

Materials necessary to collect cells:

1. Scraper–Corning cell lifter (Corning, catalog number: 3008 )
   
2. Trypsin-EDTA 0.25% (Thermo Fisher Scientific, Gibco^TM, catalog number: 25200056 )
   
3. Phosphate Buffered Saline (PBS) 1x (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010023 )
   

Equipment

Note: The brands and models indicated are the ones used by our group, similar equipment can be used as well.

1. Pipettes (10, 200, 1,000 μl)
   
2. Scissors
   
3. Graduated cylinder
   
4. Repeat pipette (Eppendorf, model: Repeater^® M4 )
   
5. Scale (Mettler-Toledo International, model: AG104 )
   
6. Microcentrifuge (Eppendorf, model: 5424 )
   
7. Homogenizer (Tissue Tearor, Bio Spec Products, catalog number: 780CL-04 )
   
8. Sonic Dismembrator (Fisher Scientific, model: Model 100 Sonic Dismembrator)
   
9. Spectrophotometer (BioTek Instruments, model: Epoch 2 )
   
10. Vortex (Scientific Industries, model: Vortex-Genie 2 , catalog number: G560)
    
11. Plate reader (Molecular Devices, model: SpectraMax Gemini XPS )

Software

1. Gen5 Microplate Reader and Imager Software (BioTek Instruments)
   
2. Microsoft Excel (Microsoft Corporation)
   
3. SoftMax Pro 6 (Molecular Devices, LLC)
   
4. GraphPad Prism 7 (GraphPad Software, Inc)

Procedure

The CD38 activity assay can be done mainly with three different sources of CD38, cells, tissues, and recombinant enzyme. We are going to describe the procedure for cells and tissues first, and then describe the procedure using recombinant CD38 enzyme. The assay is performed in a 96-well white opaque plate (refer to Notes for plate information), at least in duplicates, with a final volume of 200 μl per well.

1. Cell and tissue samples
   It is necessary to lyse the cells and tissue samples to be able to measure CD38 activity. To do so, we lyse the samples in sucrose buffer with sonication. The optimal volume of buffer should be empirically determined for each cell type and pellet size, as well as tissue weight, to ensure efficient lysis and an optimal final concentration of proteins in the lysate. We normally use 300 μl for 20 mg of tissue, and 100 μl of sucrose buffer if using cells from a 60 mm dish (2-3 x 10⁶ cells). Keep buffer and samples on ice during all the assay steps.
   1. Sample preparation
      1. Cells: wash cells in PBS, collect them according to standard procedures suitable for each cell type (adherent cells via scraping or trypsinization; cells that grow in suspension via transferring and pelleting) in 1.5 ml tubes and pellet them by centrifugation (30 sec, at 11.7 x g). Aspirate supernatant, re-suspend cells with 100 μl of sucrose buffer, if using 2-3 x 10⁶ cells) and sonicate the samples at 30-50 W of power and 20 kHz of frequency on ice, for 3 times of 5 sec each.
         
      2. Tissue: in a 2 ml tube, add the piece of tissue (approx. 20 mg), add 300 μl of sucrose buffer and homogenize with scissors and a mechanical homogenizer until there are no visible chunks. Then, sonicate as explained above for cells. Remember to keep samples on ice during this process to avoid that the heat generated degrades the CD38 enzyme.
         After sonicating, centrifuge samples for 10 min at 13.8 x g, and at 4 °C. Transfer supernatant to a new tube for protein measurement by the Bio-Rad protein assay, and following steps. Discard pellet.
   2. Normalizing samples
      Normalize samples with sucrose buffer to obtain a mass of 20-100 μg of protein if tissue is the source of enzyme, or 50-100 μg of protein per well if cells are being used. The volume of sample to be pipetted into each well is 100 μl. For tissues with high CD38 expression, like spleen, aim to the lower end of the interval. Proceed to “Prepare reaction mix” step.
   
   
2. Recombinant CD38 enzyme
   If using commercially available recombinant CD38 enzyme, the preparation of the assay differs from how it is done with cells and tissues. The total volume in the well is maintained at 200 μl, the reaction mix is the same (100 μl) but the volume of enzyme mix can vary if one wants to test an inhibitor or activator. This method can also be adapted to use with cells or tissue samples, although our group normally treat cells or animals previously with inhibitors/activators and proceed as described above.
   1. Prepare test compounds/inhibitors
      Prepare a dilution of the test compounds 4x of the desired final concentration in sucrose buffer to a volume of 50 μl per well. Also, prepare a blank sample, with 50 μl of sucrose buffer and no test compounds. We recommend the use of a known CD38 inhibitor, such as 78c (50 nM final), or anti-CD38 antibodies as a control for the CD38 activity and test compounds. For human samples or human recombinant CD38 enzyme we suggest the use of Isatuximab (1 μg/ml), a well-known monoclonal anti-CD38 antibody already in Phase 3 clinical trials for Multiple Myeloma.
      
   2. Prepare enzyme mix
      The enzyme mix, which contains Recombinant enzyme (10 ng/μl), BSA (40 mg/ml in water) and sucrose buffer, will have a total volume of 50 μl per well. We suggest an incubation period of 15 min at room temperature if antibodies are being used, based on our experience. For 78c no incubation is required, since this molecule only binds to the enzyme-substrate product. Calculate the number of wells that are going to be used and use the following proportion to prepare the enzyme mix: Recombinant enzyme 1 μl/well; BSA 4 μl/well; and sucrose buffer 45 μl/well.
   
   Note: The following steps are common for both samples type.
   3. Prepare reaction mix
      1. Based on which CD38 activity one wants to measure, two different substrates are added to sucrose buffer. For the hydrolase/NADase activity, ε-NAD is used. For the cyclase activity, NGD is the substrate of choice.
         
      2. Prepare the reaction mix based on the number of wells to be used, considering 100 μl of reaction mix per well. For each 1 milliliter of total reaction mix, 5 μl of 10 mM ε-NAD and 40 μl of 10 mM NGD should be added, in order to achieve a final concentration of 50 and 200 μM, respectively. Vortex the tube and leave it at room temperature.
   4. Setting up the reaction
      1. Before start pipetting samples to the plate, make sure that the plate reader is properly configured. Set up machine to read fluorescence at 300 nm excitation and 410 nm emission. Configure settings of plate type (96-well opaque), analysis type (kinetics), time of analysis (at least 1 h, readings every 30 sec), define area to be read on plate, and set it up to shake once for 5 sec before start reading.
         
      2. Finally, pipet 100 μl of normalized samples, or 50 μl of recombinant enzyme mix plus 50 μl of inhibitor/activator tested, at least in duplicates. It is advised to pipet sucrose buffer in a set of wells as a blank. Then, quickly add with a repeater pipette 100 μl of reaction mix to all wells, load plate on reader tray, and read.

Data analysis

Plot the values obtained in an X-Y graph. On the X-axis plot the time, and on the Y-axis plot the fluorescence unit values obtained during the reading process (Figure 3). For statistical analysis, we use GraphPad Prism. The enzymatic activity curves obtained can be compared to evaluate the effects of the compound studied in the CD38 enzyme. For instance, in Figure 3, the black curve “Buffer” indicates the enzyme function without a test compound (where sucrose buffer was added to the enzyme). The grey curve shows the effect of the CD38 inhibitor, 78c, clearly demonstrating that there was little increase in activity over time. The red and pink “Antibodies” curve shows the CD38 activity when an Anti-CD38 antibody, in two different concentrations, was added to the enzyme. The green curves represent two concentrations of an unknown test compound being evaluated for CD38 inhibition, in this case showing little effect on CD38 activity. “No enzyme” refers to wells where only sucrose buffer and reaction mix were added. Figures 3C and 3D show the raw data and graph of percentage of activity in a chosen time point for all the compounds previously described. To compare two data sets, we perform a Student’s t-test.


Figure 3. Data analysis on GraphPad Prism. A. Raw data; B. Final activity graph based on raw data analysis; C. Table of % of control values calculated per condition; D. Final % of control graph.

Notes

1. It is important that in key experiments, results are also confirmed by following the degradation of NAD⁺, the natural substrate for both the CD38 NADase and cyclase enzymes, using HPLC to follow the hydrolysis of NAD⁺ to ADPR and cADPR (Aksoy et al., 2006).
   
2. NADase activity can also be verified by HPLC analysis, performed by anion-exchange chromatography using an AG MP-1 column (Bio-Rad) eluted with a non-linear gradient of trifluoroacetic acid, as described previously (Aksoy et al., 2006). The nucleotides are detected by UV absorption at 254 nm, and the authenticity of NAD⁺ is confirmed by co-elution with NAD⁺ standards (Aksoy et al., 2006).
   
3. We utilize 96-well standard opaque white plates for our assays, but we’ve obtained similar results with black plates, although they produce lower RFU values.
   

Recipes

1. Sucrose Buffer (500 ml)
   1. Weigh 42.7875 g of sucrose and 2.4228 g of Tris base to achieve a concentration of 0.25 M Sucrose and 40 mM Tris
      
   2. Add 400 ml of Nanopure water, add the sucrose and Tris, adjust pH to 7.4 with HCl and complete the volume to 500 ml in a graduated cylinder
      
   3. Store at 4 °C up to 6 months
2. rhCD38 enzyme buffer (5 ml)
   1. Weigh 24.4 mg of MES and 43.83 mg of NaCl to achieve a concentration of 25 mM MES and 150 mM NaCl
      
   2. Add 4 ml of Nanopure water, add the MES and NaCl, adjust pH to 6.5 with HCl and complete the volume to 5 ml in a graduated cylinder

Acknowledgments

This work was supported in part by grants from the Ted Nash Long Life Foundation, the Glenn Foundation for Medical Research via the Paul F. Glenn Laboratories for the Biology of Aging at the Mayo Clinic, a grant from Calico Laboratories, the Mayo Foundation, National Institutes of Health (NIH) grants from the National Institute of Aging (NIA, grant AG-26094), the Pancreatic Cancer SPORE project from NIH/NCI to E.N.C (grant CA102701-08), the Mayo Clinic Center for Cell Signaling in Gastroenterology (NIDDK P30DK084567). Maria Auxiliadora-Martins was supported by a grant from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Brazil (#2016/20089-4).
Declaration of interests: Dr. Chini holds a patent on the use of CD38 inhibitors for metabolic diseases.

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