Un colorimetrico metodo che misura particolare Granzyme B proteolitici attività: idrolisi di 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
Granzimi sono una famiglia di serina proteasi presenti nei lisosomi secretori di natural killer (NK) e dei linfociti T citotossici (CTL) 1. Esistono cinque differenti granzimi nell'uomo (A, B, H, K e M), e dieci nei topi (A – G, K, M e N) 2,3. Granzyme A e B Granzyme (GzmA, GzmB) sono i più abbondanti e sono stati ampiamente studiati nel contesto umano e roditori.
La funzione classica GzmB è l'induzione di apoptosi nelle cellule bersaglio eseguite in combinazione con la proteina perforina formano pori, che permette di accedere granzyme citosol cellula bersaglio 4. Anche se l'espressione GzmB è inequivocabilmente trova in linfociti citotossici, studi recenti stanno affrontando una serie di altri tipi di cellule-GzmB esprimono, compreso ma non limitato ai cheratinociti 5, 6 basofili, mastociti 7, cellule dendritiche plasmacitoidi 8, e le cellule B 9, 10.In questo contesto, le funzioni GzmB non-apoptotici sono stati rivelati vanno dalla partecipazione a processi infiammatori, rimodellamento dei tessuti e altre proprietà immunomodulanti 11-14.
Dato che un ruolo biologico più ampio è stato proposto per GzmB quanto precedentemente sospettato, ricercatori richiedono strumenti affidabili e specifici per la sua individuazione. Di vantaggio è requisito specifico di GzmB per fendere sul lato carbossilico di residui di acido aspartico, una struttura unica tra serina proteasi eucariotiche 15. Mouse, umano e di ratto GzmB sono strutturalmente molto simile, tuttavia la specificità di substrato di topo GzmB estesa differisce leggermente da quella di umano e di ratto 16, il che significa che certi substrati generici con Asp al terminale (P1) possono essere scissi efficientemente GzmB da tutte e tre le specie, mentre altri substrati con sequenze più complesse monte di P1 può dare risultati molto variabili. Sia nel passato e li più recenteterature, questo fatto ha provocato una notevole confusione e cattiva interpretazione del significato biologico di alcuni risultati sperimentali, anche se attentamente controllato, studi cinetici hanno cercato di correggere la situazione 17.
In questo articolo abbiamo cercato di illustrare questi punti con due substrati disponibili in commercio, vale a dire Boc-Ala-Ala-Asp-SBzl e N-acetil-Ile-Glu-Pro-Asp-p-nitroanilide. I due reagenti fare generare diversi gruppi reattivi a seguito di scissione (un sulfidrilico gratuito contro un paranitroanilide gratuito fluorescente), ma questo non ha alcun effetto su qualunque taglio proteolitico. Il protocollo descritto è un adattamento moderno di un protocollo molto vecchio 18, ma dovrebbe aiutare gli investigatori di utilizzare i diversi substrati GzmB adeguatamente, fornendo inoltre un quadro metodologico per rilevare l'attività di altri granzimi, come GzmA e GzmH.
Protocol
Representative Results
Discussion
Storicamente i granzimi sono stati identificati come molecole effettrici chiave di linfociti citotossici (cellule CTL e NK) in grado di indurre una rapida morte apoptotica nelle cellule bersaglio. Questo è principalmente dovuto all'azione di GzmB che scisso molecole substrato bersaglio a (D) residui di aspartato e quindi era in grado di attivare la cascata caspasi da entrambi da tagliare pro-caspasi, così come molti dei loro bersagli a valle. Tuttavia, è ora apprezzato che l'espressione GzmB non è limitato a…
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 |
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