Een colorimetrische test waarmee specifiek Granzyme B proteolytische activiteit: Hydrolyse van 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
Granzymen zijn een familie van serine proteasen in de secretoire lysosomen van natural killer (NK) cellen en cytotoxische T-lymfocyten (CTL) 1. Vijf verschillende granzymen aanwezig in mensen (A, B, H, K en M) en tien muizen (A – G, K, M en N) 2,3. Granzyme A en Granzyme B (GzmA, GzmB) zijn de meest voorkomende en zijn uitgebreid onderzocht in de mens en knaagdieren setting.
De klassieke functie van GzmB is de inductie van apoptose in doelcellen uitgevoerd in samenhang met de porievormende eiwit perforin, die granzyme om de doelcel cytosol 4 toelaat. Hoewel GzmB expressie ondubbelzinnig in cytotoxische lymfocyten, hebben recente studies over uiteenlopende andere GzmB expressie celtypen, waaronder maar niet beperkt tot keratinocyten 5, 6 basofielen, mestcellen 7, plasmacytoïde dendritische cellen 8, B-cellen en 9, 10.In dit verband, werden niet-apoptotische GzmB functies geopenbaard variërend van deelname ontstekingsprocessen, weefsel remodellering en andere immuunregulerende eigenschappen 11-14.
Aangezien een breder biologische rol is voorgesteld voor GzmB dan vroeger werd gedacht, onderzoekers vereisen betrouwbare en specifieke instrumenten voor de detectie. Van voordeel is GzmB's specifieke eis aan te hangen aan de car- kant van asparaginezuurresiduen, een eigenschap uniek onder eukaryote serineproteases 15. Muis, mens en rat GzmB structureel zeer vergelijkbaar, maar de uitgebreide substraat specificiteit van muis GzmB subtiel verschilt van die van menselijke en rat 16, hetgeen betekent dat bepaalde algemene substraten met een Asp in de terminal (P1) efficiënt kan worden gesplitst door GzmB van alle drie de soorten, terwijl andere substraten met meer gecompliceerde sequenties stroomopwaarts van P1 kunnen sterk wisselende resultaten geven. Zowel in het verleden en meer recente literature, dit feit aanzienlijke verwarring en misinterpretatie van de biologische betekenis van sommige experimentele resultaten veroorzaakt, hoewel zorgvuldig gecontroleerde hebben kinetische studies getracht om de situatie te 17 corrigeren.
In dit document hebben wij getracht deze punten met twee commercieel beschikbare substraten, namelijk Boc-Ala-Ala-Asp-SBzl en N-acetyl-Ile-Glu-Pro-Asp-p-nitroanilide illustreren. De twee reagentia genereren verschillende reactieve groepen na splitsing (vrije sulfhydrylgroep versus een fluorescerende vrij paranitroanilide), maar dit heeft geen gevolg van proteolytische splitsing. De beschreven protocol is een modern aanpassing van een zeer oud protocol 18, maar moet helpen onderzoekers verschillende substraten GzmB passende wijze gebruik en ook een methodologisch kader voor het detecteren van de activiteit van andere granzymen, zoals GzmA en GzmH.
Protocol
Representative Results
Discussion
Historisch gezien granzymen werden geïdentificeerd als belangrijke effector moleculen van cytotoxische lymfocyten (CTL en NK cellen) kan induceren een snelle apoptotische dood in doelwitcellen. Dit was voornamelijk te wijten aan de werking van GzmB, waarvan bedoelde substraat moleculen gesplitst op aspartaat (D) residuen en derhalve kon de caspase cascade activeert zowel splitsen pro-caspasen, evenals verscheidene hun stroomafwaartse doelwitten. Het is echter nu duidelijk zijn dat GzmB expressie niet beperkt tot lymfo?…
Divulgations
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. Biochimie. 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).
