A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

Summary

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

Abstract

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB’s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

Introduction

Granzymes are a family of serine proteases found in the secretory lysosomes of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) ¹. Five different granzymes exist in humans (A, B, H, K and M), and ten in mice (A - G, K, M and N) ^2,3. Granzyme A and Granzyme B (GzmA, GzmB) are the most abundant and have been extensively investigated in the human and rodent setting.

The classic function of GzmB is the induction of apoptosis in target cells executed in conjunction with the pore-forming protein perforin, which permits the granzyme to access the target cell cytosol ⁴. Although GzmB expression is unequivocally found in cytotoxic lymphocytes, recent studies have been addressing a variety of other GzmB-expressing cell types, including but not limited to keratinocytes ⁵, basophils ⁶, mast cells ⁷, plasmacytoid dendritic cells ⁸, and B cells ^9,10. In this context, non-apoptotic GzmB functions were revealed ranging from participation in inflammatory processes, tissue remodelling and other immunoregulatory properties ^11-14.

Given that a broader biological role has been proposed for GzmB than previously suspected, researchers require reliable and specific tools for its detection. Of advantage is GzmB’s specific requirement to cleave on the carboxyl side of aspartic acid residues, a property unique among eukaryotic serine proteases ¹⁵. Mouse, human and rat GzmB are structurally very similar, however the extended substrate specificity of mouse GzmB differs subtly from that of both human and rat ¹⁶, which means that certain generic substrates with Asp at the terminal (P1) can be cleaved efficiently by GzmB from all three species, whereas other substrates with more complicated sequences upstream of P1 may give widely variable results. In both the past and more recent literature, this fact has caused considerable confusion and misinterpretation of the biological significance of some experimental findings, even though carefully controlled, kinetic studies have sought to correct the situation ¹⁷.

In this paper we have sought to illustrate these points using two commercially available substrates, namely Boc-Ala-Ala-Asp-SBzl and N-acetyl-Ile-Glu-Pro-Asp-p-nitroanilide. The two reagents do generate different reactive groups following cleavage (a free sulphydryl versus a fluorescent free paranitroanilide), but this has no effect whatever on proteolytic cleavage. The described protocol is a modern adaption of a very old protocol ¹⁸, but should help investigators to use the different GzmB substrates appropriately, while also providing a methodological framework for detecting the activity of other granzymes, such as GzmA and GzmH.

Protocol

NOTE: Spleens were derived from mice (6-10 weeks of age) and all animal experiments were performed according to the animal ethics guidelines of the Peter MacCallum Cancer Centre (E486).

1. Preparation of Samples.

1. Preparation of activated mouse NK cells.
   1. Isolate primary naive NK cells from single-cell suspensions of the spleens of C57BL/6 or B6.GrzmB-/- (GzmB gene null) mice by negative selection using commercially available kits.
   2. Follow the manufacturers’ protocol. Briefly, magnetically label “non-NK” cells such as T and B cells, dendritic cells, granulocytes, macrophages, and red blood cells using biotin-conjugated antibodies against their specific surface markers. Use magnetic separation with anti-Biotin beads to deplete “non-NK” cells.
   3. Culture isolated NK cells for 5-7 days in media containing IL-2 (1,000 U/ml) at 700,000 cells/ml in 24-well plates at 37 °C.
   4. Alternatively, use activated T cells ¹⁹, primary antigen-restricted CTL from OT1 T cell receptor transgenic mice ²⁰, CTL generated in mixed lymphocyte cultures ²¹, or supernatants from activated NK cells to determine the activity of secreted GzmB ¹⁹.
2. Maintenance of human NK cell line and isolation of primary NK cells.
   1. Maintain human NK leukemia (YT cells) in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal calf serum. Alternatively, purify primary NK cells from the peripheral blood of healthy subjects and stimulate for 2-3 days in medium containing IL-2 at 100 U/ml as previously described ²².
3. Generation of cell lysates.
   1. Wash cells three times in PBS (pH 7.4), then suspend in Nonidet P-40 lysis buffer (0.1% NP-40, 250 mM NaCl, 25 mM Hepes, 2.5 mM EDTA). Ideally, lyse a minimum of 5 x 10⁶ NK cells, or 1 x 10⁷ T cells in 100 µl buffer. Do not add protease inhibitors, as many are irreversible inhibitors of serine proteases, and in particular tryptases such as GzmA. Incubate on ice for 20 min, and then pellet the nuclei in a microcentrifuge at 15,000 x g for 10 min. Discard the nuclear pellet and transfer the supernatant to a fresh tube.
4. Determine the protein concentration of the lysates.
   1. Use a commercially available protocol of choice such as, Bradford or bicinchoninic acid (BCA) and determine the protein concentration. Ensure that the concentration is ~2 mg/ml minimum. Store lysates at -20 °C until required (granzyme activity is stable for many months at -20 °C).

2. Granzyme B Activity Assay

1. Preparation of Reagents
   1. Prepare a 10 mM stock of the substrates (Boc-AAD-S-Bzl or Ac-IEPD-pNA) in DMSO (~5 mg/ml) and store in aliquots at -20 °C. Prepare a working dilution freshly and discard after each day, as Boc-AAD-S-Bzl partially hydrolyzes on storage in DMSO.
   2. Prepare a 250 mM stock of the colorimetric reagent (5,5’-dithio-bis(2-nitrobenzoic acid), DTNB) in DMSO (0.09 g/ml) and store at -20 °C. Prepare a fresh working dilution each day. This reagent is only required for substrates with SBzl indicator groups, not pNA.
   3. Make up fresh buffer (0.1M HEPES, 0.05 M MgCl_2, pH 7.3) every 2-3 weeks.
2. Bring DTNB and synthetic substrates to room temperature. Dilute substrates (1:16, e.g. 100 µl in 1.6 ml of buffer) and DTNB (1:250, e.g. 10 µl in 2.5 ml buffer) and mix well.
3. Dilute a minimum of 100 µg protein lysate of each sample in buffer to a final volume of 160 µl. Using a multichannel pipette add 50 µl per well in triplicate (~33.3 µg/well) in a ‘‘U’’ bottom vinyl 96-well plate. Include a “buffer-only” control (also in triplicate).
4. Add 100 µl of diluted DTNB to the samples/buffer control (omit this step when working with Ac-IEPD-pNA.
   NOTE: Cleavage of Ac-IEPD-pNA by GzmB directly releases fluorogenic pNA (Figure 4B).
5. Use a multichannel pipette to quickly add 50 µl of diluted substrates to each set of triplicates, starting with the buffer then any negative controls and finishing with expected positive samples.
6. Place the plate in the plate reader. Measure Asp-ase activity by hydrolysis of Boc-Ala-Ala-Asp-S-Bzl/Ac-IEPD-pNA in a microplate reader at OD 405 nm. Use a kinetic assay protocol, take 25 readings, every 15 sec (ca. 6 min total reading time).
7. Use the absorbance readings generated at each time point to manually calculate the rate of substrate cleavage if the plate-reader’s software cannot generate a value for Vmax.

3. Analysis of Data

NOTE: The data generated is presented as maximum velocity, defined as change of OD over time (mOD/min).

1. To analyze the data, determine the maximum rate of substrate cleavage, which is calculated from the rate of change in absorbance. Do this automatically using the plate reader software, which typically has this option.
2. Alternatively after viewing the kinetic plots of change in absorbance, which are generated during the kinetic assay on the plate reader, manually select the data points which give the steepest straight line. For most assays the maximum rate is achieved within the first 2 min of the assay.
3. Subtract the initial OD reading from the highest OD reading on this straight line and divide by the time interval. Transfer raw data to Microsoft Excel/Graph pad for statistical analysis and graphic display.

Results

The Boc-AAD-S-Bzl substrate is specific for GzmB

The serine protease GzmB is a major constituent of cytotoxic lymphocytes (CTL and NK cells) and is predominantly responsible for inducing rapid apoptotic death in target cells, such as virus-infected or transformed cells. This is largely due to its substrate preference for cleavage after certain specific aspartate residues in selected proteins, an attribute shared with the caspases, which also cleave after aspartate residues but...

Discussion

Historically the granzymes were identified as key effector molecules of cytotoxic lymphocytes (CTL and NK cells) capable of inducing a rapid apoptotic death in target cells. This was principally due to the action of GzmB, which cleaved target substrate molecules at aspartate (D) residues and thus was able to activate the caspase cascade by both cleaving pro-caspases, as well as several of their downstream targets. However, it is now appreciated that GzmB expression is not confined to lymphoid cytotoxic cells and its func...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Boc-Ala-Ala-Asp-S-Bzl	SM Biochemicals LLC, CA	SMSB05	Granzyme B substrate (mouse and human)
Ac-IEPD-pNA	SM Biochemicals LLC, CA	SMPNA009	Granzyme B substrate (only human)
N-α-CBZ-L-lysine-S-Bzl	Sigma-Aldrich	C3647	Granzyme A substrate
Suc-Phe-Leu-Phe-S-Bzl	SM Biochemicals LLC, CA	SB025	Granzyme H substrate
5,5’-dithio-bis(2-nitrobenzoic acid)	Sigma-Aldrich	D8130	DTNB, Ellman’s Reagent
NK cell isolation kit II mouse	Miltenyi Biotec GmbH	130-096-892	negative selection kit
NK cell isolation kit human	Miltenyi Biotec GmbH	130-092-657	negative selection kit
Plate reader	Biorad iMark		Biorad Microplate Manager Software Version MPM6.3
Serocluster U-bottom vinyl 96-well plate	Corning, MA, USA	2797