Um ensaio colorimétrico que mede Especificamente granzima B Actividade proteolítica: Hidrólise de 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
Granzimas são uma família de serina-proteases encontradas nos lisossomas secretoras de células assassinas naturais (NK) e linfócitos T citotóxicos (CTL) 1. Cinco granzimas diferentes existem em seres humanos (A, B, H, K e M), e dez ratos (A – G, K, M e N) 2,3. Granzyme A e B Granzyme (GzmA, GzmB) são os mais abundantes e têm sido amplamente estudados no cenário humano e roedores.
A função clássica de GzmB é a indução de apoptose em células alvo executadas em conjunto com a proteína perforina de formação de poros, o que permite a granzima para aceder ao citosol da célula alvo 4. Embora a expressão GzmB é inequivocamente encontrada em linfócitos citotóxicos, os estudos recentes têm-se debruçado sobre uma variedade de outros tipos de células que expressam GzmB, incluindo mas não se limitando aos queratinócitos 5, 6 basófilos, mastócitos, células dendríticas 7 plasmocitoides 8, 9 e células B, 10.Neste contexto, as funções GzmB não apoptóticas foram revelados desde a participação em processos inflamatórios, remodelação de tecidos e outras propriedades imunomoduladoras 11-14.
Tendo em conta que um papel biológico mais amplo tem sido proposto para GzmB do que se suspeitava anteriormente, os pesquisadores necessitam de ferramentas confiáveis e específicos para a sua detecção. De vantagem é requisito específico da GzmB para clivar no lado carboxilo dos resíduos de ácido aspártico, uma propriedade únicas entre as proteases de serina 15 eucarióticas. Rato, humano e de rato GzmB são estruturalmente muito semelhantes, no entanto, a especificidade do substrato prolongado de rato GzmB subtilmente diferente daquele de ambos humano e de rato 16, o que significa que certos substratos genéricos com Asp no terminal (P1) pode ser clivado eficazmente por GzmB de todas as três espécies, enquanto que outros substratos com sequências mais complicadas montante de P1 pode dar resultados muito variáveis. Tanto no passado e li mais recenteterature, este fato tem causado confusão e má interpretação do significado biológico de alguns achados experimentais considerável, embora cuidadosamente controlados, estudos cinéticos têm procurado corrigir a situação 17.
Neste trabalho procuramos ilustrar esses pontos usando dois substratos disponíveis no mercado, ou seja, Boc-Ala-Ala-Asp-SBzl e N-acetil-Ile-Glu-Pro-Asp-p-nitroanilida. Os dois reagentes fazer gerar diferentes grupos reativos após a clivagem (a sulfidrila livre versus um paranitroanilida livre fluorescente), mas isso não tem nenhum efeito sobre a clivagem proteolítica. O protocolo descrito é uma adaptação moderna de um protocolo muito velho 18, mas deve ajudar os pesquisadores a utilizar os diferentes substratos GzmB adequadamente, além de fornecer um quadro metodológico para detectar a atividade de outros granzimas, como GzmA e GzmH.
Protocol
Representative Results
Discussion
Historicamente os granzimas foram identificados como moléculas efectoras principais de linfócitos citotóxicos (CTL e as células NK), capazes de induzir uma rápida morte apoptótica em células alvo. Isto foi devido principalmente à ação de GzmB, que clivada moléculas de substrato-alvo em aspartato (D) os resíduos e, assim, foi capaz de ativar a cascata caspase por ambas clivagem pró-caspases, assim como vários de seus alvos a jusante. No entanto, é agora apreciado que a expressão GzmB não está confinada …
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 |
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