Et kolorimetrisk assay, der specifikt måler Granzyme B proteolytisk aktivitet: Hydrolyse af 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
Granzymer er en familie af serinproteaser fundet i sekretoriske lysosomer af naturlige dræber (NK) celler og cytotoksiske T-lymfocytter (CTL) 1. Der er fem forskellige granzymer i mennesker (A, B, H, K og M), og ti mus (A – G, K, M og N) 2,3. Granzyme A og Granzyme B (GzmA, GzmB) er den mest rigelige og er blevet grundigt undersøgt i det menneskelige og gnaver indstilling.
Den klassiske funktion GzmB er induktion af apoptose i målceller udført i forbindelse med poredannende protein perforin, som tillader granzym adgang til målcellen cytosol 4. Selv GzmB ekspression utvetydigt findes i cytotoksiske lymfocytter, har nylige studier beskæftiget sig en række andre GzmB-udtrykkende celletyper, herunder, men ikke begrænset til keratinocytter 5, basofiler 6, mastceller 7, plasmacytoide dendritiske celler 8, og B-celler 9, 10.I den forbindelse blev der ikke apoptotiske GzmB funktioner afsløret lige fra deltagelse i inflammatoriske processer, vævsombygning og andre immunregulerende egenskaber 11-14.
Eftersom der er blevet foreslået en bredere biologiske rolle for GzmB end tidligere formodet, forskerne have pålidelige og konkrete værktøjer til sin opdagelse. Af fordel er GzmB specifikke krav om at spalte på carboxylsiden af asparaginsyrerester, en ejendom unik blandt eukaryote serinproteaser 15. Mus, human og rotte GzmB er strukturelt meget ens, men den udvidede substratspecificitet af muse GzmB adskiller diskret fra både human og rotte 16, hvilket betyder, at visse generiske substrater med Asp i terminalen (P1) kan spaltes effektivt af GzmB fra alle tre arter, kan mens andre substrater med mere komplicerede sekvenser opstrøms af P1 giver vidt forskellige resultater. I både fortiden og nyere literature har denne kendsgerning forårsaget betydelig forvirring og fejlfortolkning af den biologiske betydning af nogle eksperimentelle resultater, selv om nøje kontrolleret, har kinetiske undersøgelser forsøgt at rette op på situationen 17.
I dette papir har vi forsøgt at illustrere disse punkter ved hjælp af to kommercielt tilgængelige substrater, nemlig Boc-Ala-Ala-Asp-SBzl og N-acetyl-Ile-Glu-Pro-Asp-p-nitroanilid. De to reagenser gøre generere forskellige reaktive grupper efter spaltning (en gratis sulphydryl versus en fluorescerende gratis paranitroanilid), men dette har ingen indvirkning på proteolytisk spaltning. Den beskrevne protokol er en moderne tilpasning af en meget gammel protokol 18, men skal hjælpe efterforskerne til at bruge de forskellige GzmB substrater passende, og giver samtidig en metodologisk ramme til påvisning af aktiviteten af andre granzymer såsom GzmA og GzmH.
Protocol
Representative Results
Discussion
Historisk set granzymer blev identificeret som vigtige effektor molekyler af cytotoksiske lymfocytter (CTL og NK-celler) der er i stand til at inducere en hurtig apoptotisk død i målceller. Det var hovedsageligt på grund af virkningen af GzmB, som spaltede målsubstratet molekyler på aspartat (D) restprodukter og dermed var i stand til at aktivere caspasekaskaden af begge spalter pro-caspaser, samt flere af deres downstream mål. Imidlertid er det nu klart, at GzmB ekspression ikke er begrænset til lymf…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work received support through grant HA 6136/1-1 from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) to MH. JAT is supported by Program and Project Grants from the National Health and Medical Research Council of Australia.
Materials
| Product | Company | Catalogue number | Comment/description |
| 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-a-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 |
References
- Trapani, J. A., Browne, K. A., Dawson, M., Smyth, M. J. Immunopurification of functional Asp-ase (natural killer cell granzyme B) using a monoclonal antibody). Biochem Biophys Res Commun. 195 (2), 910-920 (1993).
- Ewen, C. L., Kane, K. P., Bleackley, R. C. A quarter century of granzymes. Cell Death Differ. 19 (1), 28-35 (2012).
- Hoves, S., Trapani, J. A., Voskoboinik, I. The battlefield of perforin/granzyme cell death pathways. J Leukoc Biol. , (2009).
- Peters, P. J., et al. Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes. J Exp Med. 173 (5), 1099-1109 (1991).
- Berthou, C., et al. Acquisition of granzyme B and Fas ligand proteins by human keratinocytes contributes to epidermal cell defense. J Immunol. 159 (11), 5293-5300 (1997).
- Tschopp, C. M., et al. Granzyme B, a novel mediator of allergic inflammation: its induction and release in blood basophils and human asthma. Blood. 108 (7), 2290-2299 (2006).
- Strik, M. C., et al. Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation. Mol Immunol. 44 (14), 3462-3472 (2007).
- Jahrsdorfer, B., et al. Granzyme B produced by human plasmacytoid dendritic cells suppresses T cell expansion. Blood. , (2009).
- Hagn, M., et al. Human B cells secrete granzyme B when recognizing viral antigens in the context of the acute phase cytokine IL-21. J Immunol. 183 (3), 1838-1845 (2009).
- Hagn, M., et al. Human B cells differentiate into granzyme B-secreting cytotoxic B lymphocytes upon incomplete T-cell help. Immunol Cell Biol. 90 (4), 457-467 (2012).
- Froelich, C. J., Pardo, J., Simon, M. M. Granule-associated serine proteases: granzymes might not just be killer proteases. Trends Immunol. 30 (3), 117-123 (2009).
- Hagn, M., Jahrsdorfer, B. Why do human B cells secrete granzyme B? Insights into a novel B-cell differentiation pathway. Oncoimmunology. 1 (8), 1368-1375 (2012).
- Susanto, O., Trapani, J. A., Brasacchio, D. Controversies in granzyme biology. Tissue Antigens. 80 (6), 477-487 (2012).
- Walch, M., et al. Cytotoxic cells kill intracellular bacteria through granulysin-mediated delivery of granzymes. Cell. 157 (6), 1309-1323 (2014).
- Odake, S., et al. Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins. Biochemistry. 30 (8), 2217-2227 (1991).
- Casciola-Rosen, L., et al. Mouse and human granzyme B have distinct tetrapeptide specificities and abilities to recruit the bid pathway. J Biol Chem. 282 (7), 4545-4552 (2007).
- Kaiserman, D., et al. The major human and mouse granzymes are structurally and functionally divergent. J Cell Biol. 175 (4), 619-630 (2006).
- Powers, J. C., Kam, C. M. Peptide thioester substrates for serine peptidases and metalloendopeptidases. Methods Enzymol. 248, 3-18 (1995).
- Hagn, M., et al. Activated mouse B cells lack expression of granzyme. B. J Immunol. 188 (2), 3886-3892 (2012).
- Konjar, S., et al. Human and mouse perforin are processed in part through cleavage by the lysosomal cysteine proteinase cathepsin L. Immunology. 131 (2), 257-267 (2010).
- Sutton, V. R., et al. Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis. J Cell Biol. 176 (4), 425-433 (2007).
- Trapani, J. A., Smyth, M. J., Apostolidis, V. A., Dawson, M., Browne, K. A. Granule serine proteases are normal nuclear constituents of natural killer cells. J Biol Chem. 269 (28), 18359-18365 (1994).
- Cullen, S. P., Adrain, C., Luthi, A. U., Duriez, P. J., Martin, S. J. Human and murine granzyme B exhibit divergent substrate preferences. J Cell Biol. 176 (4), 435-444 (2007).
- Thornberry, N. A., et al. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis. J Biol Chem. 272 (29), 17907-17911 (1997).
- Bird, C. H., et al. Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway. Mol Cell Biol. 18 (11), 6387-6398 (1998).
- Bots, M., Medema, J. P. Serpins in T cell immunity. J Leukoc Biol. 84 (5), 1238-1247 (2008).
- Sun, J., et al. A new family of 10 murine ovalbumin serpins includes two homologs of proteinase inhibitor 8 and two homologs of the granzyme B inhibitor (proteinase inhibitor 9). J Biol Chem. 272 (24), 15434-15441 (1997).
- Bird, C. H., Hitchen, C., Prescott, M., Harper, I., Bird, P. I. Immunodetection of granzyme B tissue distribution and cellular localisation. Methods Mol Biol. 844, 237-250 (2012).
- Jenkins, M. R., et al. Visualizing CTL activity for different CD8+ effector T cells supports the idea that lower TCR/epitope avidity may be advantageous for target cell killing. Cell Death Differ. 16 (4), 537-542 (2009).
- Edwards, K. M., Kam, C. M., Powers, J. C., Trapani, J. A. The human cytotoxic T cell granule serine protease granzyme H has chymotrypsin-like (chymase) activity and is taken up into cytoplasmic vesicles reminiscent of granzyme B-containing endosomes. J Biol Chem. 274 (43), 30468-30473 (1999).
- Sutton, V. R., et al. Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation. J Exp Med. 192 (10), 1403-1414 (2000).
