比色测定的具体措施颗粒酶B蛋白水解活动：水解的Boc-丙氨酸 - 丙氨酸-ASP-S-BZL的
Özet
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
粒酶是一类在自然杀伤（NK）细胞和细胞毒性T淋巴细胞（CTL）的1的分泌溶酶体发现丝氨酸蛋白酶。在人类中（A，B，H，K和M）存在五种不同的颗粒酶和10小鼠（A – G，K，M和N）2,3。颗粒酶A和颗粒酶B（GzmA，GZMB）是最丰富的，并已被广泛研究，在人类和啮齿类动物的设置。
GZMB经典功能是细胞凋亡的诱导在与孔形成蛋白穿孔，其允许粒酶访问目标细胞胞质溶胶4一起执行靶细胞。虽然GZMB表达在细胞毒性淋巴细胞明确发现，最近的研究已经解决了各种其他GZMB表达的细胞类型，包括但不限于角质细胞5，嗜碱性粒细胞6，肥大细胞7，浆细胞样树突细胞8，和B细胞9， 10。在这种情况下，非凋亡GZMB功能进行了揭示，从参与炎症过程，组织重塑和其他免疫调节性质11-14。
鉴于一个更广泛的生物作用已被提议用于GZMB比先前怀疑，研究人员需要针对其检测可靠和专用工具。的优点是GZMB的具体要求来切割上的天冬氨酸残基，一个属性之间的真核丝氨酸蛋白酶15独特的羧基侧。小鼠，人和大鼠GZMB在结构上非常相似，但是小鼠GZMB的扩展的底物特异性不同巧妙地从人类和鼠16，这意味着某些通用基板用Asp在终端（P1）可以通过GZMB有效切割的所有三个品种，而其他底材具有更复杂的序列上游P1可能会给广泛不同的结果。在过去和最近的立terature，这一事实造成了相当大的混乱和一些实验结果的生物学意义的曲解，即使严格控制的，动力学研究试图纠正这种情况17。
在本文中，我们已经设法示出了这些点使用两个市售底物，即的Boc – 丙氨酸 – 丙氨酸 – 天冬氨酸 – SBzl和N-乙酰基异亮氨酸 – 谷氨酸 – 亲天冬氨酸 – 对硝基苯胺。的两种试剂做产生不同的反应基团以下裂解（游离巯与荧光游离对硝基酰苯胺），但这种不具有任何对蛋白水解裂解的影响。所描述的协议是一个非常古老的协议，18现代适应，但应该帮助调查人员适当地使用不同的GZMB基板，同时也提供了一个方法框架，用于检测其他颗粒酶，如GzmA和GzmH的活动。
Protocol
Representative Results
Discussion
在历史上，粒酶被鉴定为细胞毒性淋巴细胞（CTL和NK细胞）能够诱导快速的凋亡性死亡的靶细胞的关键效应分子。这主要是由于GZMB的动作，这裂解靶底物分子在天门冬氨酸（D）残基，因而能由这两种裂解亲胱天蛋白酶激活的胱天蛋白酶级联，以及若干的其下游目标。然而，现在认识到，GZMB表达并不局限于淋巴细胞毒性细胞，其功能可被扩展远远超出靶细胞识别和细胞死亡。
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Açıklamalar
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 |
Referanslar
- 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. Biyokimya. 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. İmmünoloji. 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).
