Boc-Ala-Ala-Asp-S-Bzl hidrolizi, A Kolorimetrik Özellikle Granzim B proteolitik aktivitesini ölçer Deneyi
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 doğal katil (NK) hücreler ve sitotoksik T lenfositler (CTL), 1 salgılama lizozomlarında bulunan serin proteaz ailesidir. Beş farklı granzymes insanlarda (A, B, H, K ve M,) 'de bulunan, ve farelerde on (A – G, K, M ve N) 2,3. Granzim A ve Granzim B (GzmA, GzmB) en bol miktarda bulunan ve geniş ölçüde insan ve kemirgen ortamda araştırılmıştır.
GzmB klasik fonksiyonu hedef hücre sitosol 4 ulaşmak için Granzim izin gözenek oluşturucu protein perforin ile bağlantılı olarak yürütülen, hedef hücrelerde apoptoz endüksiyonu. GzmB sentezleme tümden sitotoksik lenfositlerinin de bulunmasına rağmen, son zamanlarda yapılan çalışmalar bazofiller 6, mast hücreleri 7, plazmasitoid dendritik hücreler 8 ve B hücreleri 9, aşağıdakileri içeren, ancak keratinositler 5 ile sınırlı değildir, başka bir GzmB ifade eden hücre tipleri, çeşitli ele edilmiştir, 10.Bu bağlamda, apoptotik olmayan GzmB fonksiyonları, enflamatuar süreçler, doku tekrar modellemesinde ve diğer bağışıklık düzenleyici özelliklerinin 11-14 katılım kadar ortaya çıkmıştır.
Daha geniş bir biyolojik rolü, daha önce şüphe daha GzmB önerilmiştir göz önüne alındığında, araştırmacılar da tespiti için güvenilir ve özel araçlar gerektirir. Avantajlı olarak ökaryotik serin proteazları 15 arasında benzersiz aspartik asit kalıntısı, bir özelliği karboksil yanı üzerinde bölmek için GzmB spesifik bir gerekliliktir. Fare, insan ve sıçan GzmB ancak fare GzmB uzun alt-tabaka özgüllüğü terminali (P1) Asp, bazı genel alt-tabakalar GzmB tarafından etkin bir şekilde klivaj edilebilir olduğu anlamına gelir, hem insan ve fare 16, bu ustaca farklıdır, yapısal olarak çok benzer Üç türlerinden, üst baş P1 daha karmaşık dizileri ile diğer alt-tabakalara ise, geniş çapta değişkendir sonuçlar verebilir. Geçmişte ve daha yeni li hem deterature, bu gerçeği dikkatle kontrol olsa, önemli karışıklık ve bazı deneysel bulguların biyolojik önemi yanlış yorumlanmasına neden oldu, kinetik çalışmalar durumu 17 düzeltmek için çalışmışlardır.
Bu yazıda, iki ticari olarak temin edilebilen alt tabakalar, yani, Boc-Ala-Ala-Asp-SBzl ve N-asetil-Ile-Glu-Pro-Asp-p-nitroanilid kullanılarak, bu noktaları göstermek için çalışmışlardır. İki reaktifler bölünme (bir floresan serbest paranitroanilide karşı serbest sülfidril) Aşağıdaki farklı reaktif gruplar oluşturmak, ama bu proteolitik bölünme üzerinde hiçbir etkisi ne olursa olsun sahip. Açıklanan protokol, çok eski bir protokol 18 modern bir uyarlamasıdır, fakat, aynı zamanda, GzmA ve GzmH gibi diğer granzymes aktivitesini tespit etmek için bir yöntem çerçeve sağlarken araştırmacılar, uygun farklı GzmB alt tabakaları kullanır yardımcı olacaktır.
Protocol
Representative Results
Discussion
Tarihsel olarak granzymes hedef hücreler hızlı bir apoptotik ölümünü indükleyebilen sitotoksik lenfositleri (CTL ve NK hücreleri) anahtar etkeni moleküller olarak tespit edilmiştir. Bu, aspartat (D) artıklarının hedef alt-tabaka molekülleri ayrılır ve böylece aşağı doğru hedeflerin çeşitli yanı sıra her ikisi de yaran ön-kaspaz kaspaz kaskadını aktive mümkün GzmB aksiyonu için esas oldu. Bununla birlikte, şu anda GzmB sentezleme lenfoid sitotoksik hücreler ile sınırlı değildir ve bu…
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. 生物化学. 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).
