Summary

Et kolorimetrisk assay, der specifikt måler Granzyme B proteolytisk aktivitet: Hydrolyse af Boc-Ala-Ala-Asp-S-Bzl

Published: November 28, 2014
doi:

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

Granzymer er en familie af serinproteaser fundet i sekretoriske lysosomer af naturlige dræber (NK) celler og cytotoksiske T-lymfocytter (CTL) 1. Der er fem forskellige granzymer i mennesker (A, B, H, K og M), og ti mus (A – G, K, M og N) 2,3. Granzyme A og Granzyme B (GzmA, GzmB) er den mest rigelige og er blevet grundigt undersøgt i det menneskelige og gnaver indstilling.

Den klassiske funktion GzmB er induktion af apoptose i målceller udført i forbindelse med poredannende protein perforin, som tillader granzym adgang til målcellen cytosol 4. Selv GzmB ekspression utvetydigt findes i cytotoksiske lymfocytter, har nylige studier beskæftiget sig en række andre GzmB-udtrykkende celletyper, herunder, men ikke begrænset til keratinocytter 5, basofiler 6, mastceller 7, plasmacytoide dendritiske celler 8, og B-celler 9, 10.I den forbindelse blev der ikke apoptotiske GzmB funktioner afsløret lige fra deltagelse i inflammatoriske processer, vævsombygning og andre immunregulerende egenskaber 11-14.

Eftersom der er blevet foreslået en bredere biologiske rolle for GzmB end tidligere formodet, forskerne have pålidelige og konkrete værktøjer til sin opdagelse. Af fordel er GzmB specifikke krav om at spalte på carboxylsiden af asparaginsyrerester, en ejendom unik blandt eukaryote serinproteaser 15. Mus, human og rotte GzmB er strukturelt meget ens, men den udvidede substratspecificitet af muse GzmB adskiller diskret fra både human og rotte 16, hvilket betyder, at visse generiske substrater med Asp i terminalen (P1) kan spaltes effektivt af GzmB fra alle tre arter, kan mens andre substrater med mere komplicerede sekvenser opstrøms af P1 giver vidt forskellige resultater. I både fortiden og nyere literature har denne kendsgerning forårsaget betydelig forvirring og fejlfortolkning af den biologiske betydning af nogle eksperimentelle resultater, selv om nøje kontrolleret, har kinetiske undersøgelser forsøgt at rette op på situationen 17.

I dette papir har vi forsøgt at illustrere disse punkter ved hjælp af to kommercielt tilgængelige substrater, nemlig Boc-Ala-Ala-Asp-SBzl og N-acetyl-Ile-Glu-Pro-Asp-p-nitroanilid. De to reagenser gøre generere forskellige reaktive grupper efter spaltning (en gratis sulphydryl versus en fluorescerende gratis paranitroanilid), men dette har ingen indvirkning på proteolytisk spaltning. Den beskrevne protokol er en moderne tilpasning af en meget gammel protokol 18, men skal hjælpe efterforskerne til at bruge de forskellige GzmB substrater passende, og giver samtidig en metodologisk ramme til påvisning af aktiviteten af andre granzymer såsom GzmA og GzmH.

Protocol

BEMÆRK: Milte blev afledt fra mus (6-10 uger gamle) og alle dyreforsøg blev udført i overensstemmelse med de dyreetiske retningslinjer for Peter MacCallum Cancer Center (E486). 1. Fremstilling af prøver. Fremstilling af aktiverede muse NK-celler. Isoler primære naive NK-celler fra encellede suspensioner af milt fra C57BL / 6 eller B6.GrzmB – / – (GzmB gen null) mus ved negativ selektion ved hjælp af kommercielt tilgængelige kits. Følg fabrikante…

Representative Results

Boc-AAD-S-Bzl substrat er specifik for GzmB Den serinprotease GzmB er en væsentlig bestanddel af cytotoksiske lymfocytter (CTL og NK-celler) og er hovedsageligt ansvarlig for at inducere hurtig apoptotisk død i målceller, såsom virusinficerede eller transformeret celler. Dette skyldes i høj grad dets substrat præference for spaltning efter bestemte aspartatrester i udvalgte proteiner, en egenskab deles med caspaserne, som også spalter efter aspartatrester men er fra e…

Discussion

Historisk set granzymer blev identificeret som vigtige effektor molekyler af cytotoksiske lymfocytter (CTL og NK-celler) der er i stand til at inducere en hurtig apoptotisk død i målceller. Det var hovedsageligt på grund af virkningen af ​​GzmB, som spaltede målsubstratet molekyler på aspartat (D) restprodukter og dermed var i stand til at aktivere caspasekaskaden af ​​begge spalter pro-caspaser, samt flere af deres downstream mål. Imidlertid er det nu klart, at GzmB ekspression ikke er begrænset til lymf…

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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Cite This Article
Hagn, M., Sutton, V. R., Trapani, J. A. A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl. J. Vis. Exp. (93), e52419, doi:10.3791/52419 (2014).

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