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. Author manuscript; available in PMC: 2021 May 11.
Published in final edited form as: Methods Mol Biol. 2021;2248:109–119. doi: 10.1007/978-1-0716-1130-2_8

In vitro physical and functional interaction assays to examine the binding of progranulin derivative Atsttrin to TNFR2 and its anti-TNFα activity

Wenyu Fu 1, Aubryanna Hettinghouse 1, Chuan-ju Liu 1,2
PMCID: PMC8112733  NIHMSID: NIHMS1699493  PMID: 33185871

Abstract

TNFα/TNFR signaling plays a critical role in the pathogenesis of various inflammatory and autoimmune diseases, and anti-TNFα therapies have been accepted as the effective approaches for treating several autoimmune diseases. Progranulin (PGRN), a multi-faced growth-factor like molecule, directly binds to TNFR1 and TNFR2, particularly to the latter with higher affinity than TNFα. PGRN derivative Atsttrin is composed of three TNFR-binding domain of PGRN and exhibits even better therapeutic effects than PGRN in several inflammatory disease models, including collagen-induced arthritis. Herein we describe the detailed methodology of using 1) ELISA-based solid phase protein-protein interaction assay to demonstrate the direct binding of Atsttrin to TNFR2 and its inhibition of TNFα/TNFR2 interaction; and 2) tartrate-resistant acid phosphatase (TRAP) staining of in vitro osteoclastogenesis to reveal the cell-based anti-TNFα activity of Atsttrin. Using the protocol described here, the investigators should be able to reproducibly detect the physical inhibition of TNFα binding to TNFR and the functional inhibition of TNFα activity by Atsttrin and various kinds of TNF inhibitors.

Keywords: TNFα, Progranulin, Atsttrin, TNFR2, ELISA-based protein-protein interaction assay, in vitro osteoclastogenesis

1. Introduction

TNFα/TNFR signaling orchestrates a wide range of inflammatory processes, and plays a crucial role in the pathogenesis of various inflammatory autoimmune diseases [1–4]. TNFR1 primarily mediates the inflammatory activity of TNFα, whereas emerging evidences indicate that TNFR2 plays a protective and anti-inflammatory role in various diseases [5–8]. Our genetic screen for the binding partners of progranulin (PGRN), a growth factor like molecule with multiple functions [9–14], led to the isolation of TNFR2 as the PGRN-binding receptor[15]. Remarkably, PGRN exhibits an approximately 600-fold higher binding affinity to TNFR2 than does TNFα. Moreover, beyond the binding of PGRN to TNFR, growing evidences demonstrate that PGRN/TNFR interactions play an important role in various kinds of diseases and conditions [12–42].

PGRN is a 593-aa secreted glycoprotein. It contains seven-and-a half repeats of a cysteine-rich motif in the order P-G-F-B-A-C-D-E and forms a unique “beads-on-a-string” structure [43,44]. In an effort to identify the minimal required domains of PGRN which retain comparable TNFR binding affinity of PGRN, an engineered protein composed of FAC domain of PGRN is created and referred to as Atsttrin (antagonist of TNFα/TNFR signaling via targeting to TNF receptors), which, similar to PGRN, exhibits higher binding affinity for TNFR2, but lower affinity for TNFR1 than TNFα [15,45,46]. More importantly, Atsttrin exhibits therapeutic effects in various diseases [15,21,34,47,48], and surpasses PGRN in collagen-induced arthritis model in vivo [15]. In addition, Atsttrin has been show to exhibit a preventive effect in non-surgically or surgically induced rodent osteoarthritis models [38], and Atsttrin-transduced mesenchymal stem cells articular treatment prevents osteoarthritis progression in surgically induced murine osteoarthritis model [49]. Atsttrin also enhances bone regeneration in several bone defect models [50]. It is known that inflammatory cytokine TNFα acts in concert with RANKL to promote osteoclastogenesis [51–54], and our previous studies demonstrated that Atsttrin inhibited osteoclastogenensis in vitro [15] and bone loss in vivo [38].

Herein, we describe the detailed methodology of using 1) ELISA based solid phase protein-protein interaction to demonstrate the direct binding of PGRN-derived Atsttrin to TNFR and its inhibition of TNFα/TNFR interaction (Fig. 1 and 2); and 2) tartrate-resistant acid phosphatase (TRAP) staining, which is widely accepted as an important cytochemical marker of oscteoclasts, to reveal the cell-based anti-TNFα activity of Atsttrin (Fig. 3).

Fig.1. ELISA based solid phase binding assay.

Fig.1

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(a) Protein to protein interaction assay to determine whether protein A binds to protein B. The immobilized protein of interest (protein A) is maximally coated into the wells of a 96 well assay plate. Unbound protein is removed by washing, and remaining surfaces are blocked with blocking buffer. The plate is washed, and binding is evaluated by incubation with serial dilutions of biotinylated interacting protein (protein B). Following another wash to remove unbound interacting protein, bound protein is detected by streptavidin-HRP and substrate development to quantify bound protein by absorbance at 450nm. (b) Inhibition binding assay to quantify the interaction between an immobilized protein (protein A) and an interacting biotinylated binding protein in the presence of varied amounts of a second binding protein of interest (protein C). All steps are conducted as in (a) with the exception that during the biotinylated protein binding step, addition of the biotinylated protein B is conducted at a constant concentration in the presence of serial concentrations of a competitive protein C.

Fig.2. Representative examples of ELISA based solid phase binding assay.

Fig.2

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(a) Binding of Atsttrin to TNFR2. (b) Atsttrin inhibition of TNFα binding to TNFR2.

Fig.3. Representative examples of TRAP staining.

Fig.3

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(a) BMDMs isolated from wild-type c57BL/6 mice were treated with M-CSF (10 ng/ml) for 3 days, then cultured with RANKL (10ng/ml) and TNFα (10 ng/ml) with or without 100ng/ml Atsttrin for 5 days and TRAP staining was performed. Scale bar, 100μm. (b) Quantitative analysis of the TRAP+ cells.

2. Materials

2.1. ELISA

  1. Recombinant proteins: Recombinant Atsttrin (Atreaon, Inc), Recombinant Human TNFR2, (R&D Systems, carrier free), Recombinant Human TNFα (R&D Systems, carrier free).

  2. EZ-Link Sulfo-NHS-Biotinylation Kit (Thermo scientific)..

  3. Zeba™ Spin Desalting Column (Thermo scientific).

  4. Tris-buffered saline (TBS): 10 mM Tris–HCl, pH 7.4, 150 mM NaCl.

  5. Binding buffer: TBS saline, 0.1 % (w/v) bovine serum albumin (BSA), 1 mM CaCl2. Prepare fresh.

  6. Streptavidin-HRP (Thermo scientific).

  7. TMB Solution (Invitrogen): Ready to use, no dilution or further preparation required. In the presence of HRP, TMB will turn to blue.

  8. Stop solution: 2 N Sulfuric Acid.

  9. Blocking solution: TBS, 5 % (w/v) bovine serum albumin (BSA).

  10. Sample diluent: TBS,0.5% BSA, 1 mM CaCl2.

  11. Costar High Binding plates (Corning).

  12. Molecular Devices plate reader

2.2. TRAP Staining

  1. Tartrate-resistant acid phosphatase (TRAP) basic incubation medium (see Note 1): 9.2g Sodium acetate anhydrous (Sigma), 7.5g L-(+)-Tartaric acid (Sigma), 950 mL distilled water, 2.8 mL Glacial acetic acid. Dissolve and adjust pH to 4.7 – 5.0 with 5M Sodium hydroxide to increase or more glacial acetic acid to decrease. Bring total volume to 1L with distilled water.

  2. Naphthol AS-MX phosphate substrate mix: 20 mg Naphthol AS-MX phosphate (Sigma), 1mL Ethylene glycol monoethyl ether (Sigma). Vortex or mix with pipette until dissolved.

  3. TRAP staining solution mix (see Note 2): 200 mL TRAP basic incubation medium, 120 mg Fast red violet LB salt (Sigma), 1mL Naphthol AS-MX phosphate substrate mix.

  4. 0.02% Fast green: 0.05g Fast green (Sigma), 250 mL Distilled water.

  5. Richard-Allan Scientific™ Cytoseal™ XYL (Thermo Scientific).

  6. Wide-type C57BL/6 mice.

  7. Dissection tools.

  8. Complete DMEM Medium: DMEM, 10% fetal bovine serum, 100U/ml penicillin, and 100μg/ml streptomycin.

  9. Osteoclast medium: DMEM, 10% fetal bovine serum, 100U/ml penicillin, 100μg/ml streptomycin, 10 ng/mL Macrophage colony stimulating factor (M-CSF), 10 ng/mL RANKL

  10. Fixing solution: 6.75 mM Citrate, 65% acetone, 3.7% formalin.

  11. Leica bright field microscope.

3. Methods

3.1. ELISA Based Solid Phase Protein-Protein Interaction to Demonstrate the Direct Binding of Atsttrin to TNFR2

  1. Coat various doses (0–0.5 μM) of Atsttrin or BSA (serving as control) in TBS buffer to Costar High Binding plates, 100 μL per well. Perform the assay using triplicates for each set of samples (see Note 3). Seal and incubate the plates overnight at 4 °C (see Note 4).

  2. The next day, discard the coating material and tap the plates dry on a paper towel. Block the plates by adding 100 μL of blocking solution to each well. Allow the solution to sit in the wells for 1 minute and then discard it. Tap the plates dry on a paper towel (see Note 5). Add a volume of 200 μL of blocking solution to each well. Seal the plates and incubate in a 25 °C incubator for 2 hours.

  3. While the plates are blocking, biotinylate TNFR2 using the EZ-Link Sulfo-NHS-Biotinylation Kit according to the manufacturer’s instruction (see Note 6). Briefly, calculate the amount of protein then add an appropriate molar ratio of biotin reagent to the protein, followed by incubation on a rotating platform for 1 hour. Then remove the excess free biotin reagent using the Zebra Desalt Spin column. You could employ HABA assay [2–(4-hydroxyazobenzene) benzoic acid] to estimate biotin incorporation (see Note 7).

  4. After 2 hours incubation, discard the blocking buffer in the plates and tap the plates dry on a paper towel. Add a volume of 300 μL of binding buffer to each well. Allow the wells about 1 minute with the binding buffer for each wash step to increase the effectiveness of the washes. After each addition, discard the solution and tap the plates dry on a paper towel. Repeat for a total of 3–5 washes.

  5. Dilute the biotin-labeled TNFR2 in binding buffer to a concentration of 1 ng/μL. Add a volume of 100 μL of TNFR2 protein solution to the appropriate wells of the plates. Seal the plates and incubate in a 37 °C incubator for one hour.

  6. Wash the plates with binding buffer to remove unbound protein. Discard the solution in the plates and dry the plates by tapping on a paper towel. Add a volume of 300 μL of binding buffer to each well three times using multichannel pipette. After each addition, discard the solution and tap dry the plates on a paper towel. Repeat for a total of 3–5 washes.

  7. Dilute Streptavidin-HRP 1:2500 in binding buffer and add 100 μL of this reagent to each well. Seal the plates and incubate at room temperature for 15 minutes.

  8. Wash the plates with binding buffer to remove unbound streptavidin-HRP. Discard the solution in the plates and tap dry the plates on a paper towel. Add a volume of 300 μL of binding buffer to each well. After each addition, discard the solution and tap dry the plates on a paper towel. Repeat for a total of 5–7 washes

  9. Add a volume of 100 μL of TMB to each well of the plate. Incubate the plates uncovered, at room temperature until a blue color is obtained, typically within 5–30min.

  10. After the desired color is developed, add a volume of 100 μL per well of 1M phosphoric acid to stop the TMB reaction.

  11. Place the plates on a plate shaker and shake at a speed of 6 rpm for 3–5 seconds to ensure complete mixing of the TMB and acid.

  12. Read the plates at 450 nm using a Molecular Devices plate reader (see Note 8) and analyze the data (see Fig. 2a).

3.2. ELISA Based Solid-Phase Protein-Protein Interaction Assay to Determine Inhibition of TNFα Binding to TNFR2 by Atsttrin

  1. Dilute TNFR2 to 0.5μg/mL with TBS. Add the diluted solutions to the appropriate wells of Costar High Binding plates, 100 μL per well. Add TBS, to the appropriate wells of the plates to serve as a negative control, 100 μL per well. Seal the plates and incubate overnight at 4 °C.

  2. The next day, discard the coating material and tap dry the plates on a paper towel. Block the plates by adding 100 μL of blocking solution to each well. Allow the solution to sit in the wells for 1 minute and then discard. Tap dry the plates on a paper towel. Add a volume of 200 μL of blocking solution to each well. Seal the plates and incubate in a 25 °C incubator for two hours.

  3. While the plates are blocking, dilute Atsttrin and BSA to 500μg/mL in Sample Diluent. For each protein, made six, 1:2 serial dilutions in Sample Diluent.

  4. After 2 hours incubation, discard the blocking buffer in the plates and tap dry the plates on a paper towel. Add a volume of 300 μL of binding buffer to each well. Allow the binding buffer to remain in the wells for about 1 minute during each wash step to increase the effectiveness of the washes. After each addition, discard the solution and tap dry the plates on a paper towel. Repeat for a total of 3–5 washes.

  5. Add a volume of 100 μL of each protein solution to the appropriate wells of the plates. Add the sample diluent to all 0 μg/mL wells, 100 μL per well. Seal the plates and incubate in a 37 °C incubator for one hour.

  6. While the plates with proteins are incubating, biotinylate TNFα as described in Subheading 3. 3, then dilute to 1 μg/mL in Sample Diluent.

  7. Add diluted TNFα to appropriate wells of the plate, 10 μL per well. Add Sample Diluent to the “No TNFα” control wells, 10 μL per well. Shake the plates for 10 seconds, and then seal and incubate in a 37 °C incubator for two hours.

  8. Follow the steps 6–12 in Subheading 2.1 (see Fig. 2b).

3.3. TRAP Staining to Determine the Anti-TNFa Activity of Atsttrin

Use bone marrow derived macrophages (BMDMs) as osteoclast progenitors (see Note 9) (56).

  1. Collect bone marrow cells from wide-type C57BL/6 mice, culture in complete DMEM and stimulate with 10 ng/ml M-CSF for 3 days.

  2. After 3 days, change the medium to osteoclast medium along with 10 ng/mL of TNFα in the absence or presence of 100 ng/mL of Atsttrin for a total of 5–7 days. Change the medium every 2 days.

  3. Hereafter, aspirate the medium from the culture well.

  4. Wash the cells gently with 1X PBS three times.

  5. Fix the cells by incubating for 90 seconds with fixing solution at room temperature and wash with 1X PBS three times.

  6. Add 1 mL of of pre-warmed TRAP stain solution into each well of the plate to be stained and incubate at 37 °C for 1 hour. Shield the plate from light (see Note 10).

  7. After 1 hour, aspirate off the TRAP staining solution, and wash the wells 3 times with pre-warmed deionized water.

  8. Counterstain the cells with 0.02% Fast Green for 1–2 minutes.

  9. Dehydrate quickly through graded alcohols, 5 seconds each, clear in Xylene and mount in Cytoseal™ XYL.

  10. Image osteoclasts using bright field microscopy. Red coloration indicates TRAP-positive cells. TRAP-positive multinucleated cells with >3 nuclei visualized by light microscopy are recorded as osteoclasts (see Fig. 3).

4. Notes

  1. Tartrate-resistant acid phosphatase (TRAP) basic incubation medium can be stored at room temperature for 6 months.

  2. Prepare TRAP staining solution mix fresh every time.

  3. It is recommended to perform the experiment in technical triplicate to get reliable results. Also, the experiments should be repeated at least three times to ensure that the results are reproducible.

  4. Although the ELISA based solid-phase protein-protein interaction assay is readily amendable to a number of different situations, a few key points must be observed to ensure success. Importantly, sodium azide must be excluded from all buffers. The inclusion of sodium azide in the buffer will inhibit the activity of HRP and can quench peroxidase activity, thus, rendering no signal.

  5. It is critical to remove any residual fluids, but do not let the plate become completely dry. When the wells are completely dry, the active components on the plate will become inactivated, which will negatively impact assay results.

  6. EZ-Link Sulfo-NHS-Biotin is moisture sensitive. Dissolve the biotin reagent immediately before use. The NHS-ester moiety readily hydrolyzes and becomes non-reactive; therefore, discard any unused reconstituted reagent.

  7. Although the biotin-based solid-phase assay offers numerous advantages over other assays, one potential disadvantage associated with this assay is that the biotinylation process may alter the structure and properties of the proteins of interest, which may also led to less or no binding activities of labeled proteins to its binding partners.

  8. Read plate immediately after adding stop solution.

  9. BMDMs are used in the protocol for osteoclast differentiation, this protocol could also be modified accordingly to apply on Raw264.7 cells. The exception is Raw264.7 cells express both M-CSF and its receptor c-fms, therefore the addition of RANKL alone is sufficient to induce osteoclast differentiation.

  10. Incubation time varies according to the amount and activity of TRAP in the samples. Stop the reaction in the appropriate stage while observation is carried out microscopically.

Acknowledgements

This work was supported partly by NIH research grants R01AR062207, R01AR061484, R01NS103931 and a DOD research grant W81XWH-16-1-0482. The recombinant Atsttrin protein is kindly provided by Atreaon, Inc.

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