En kolorimetrisk analyse som spesifikt Måler granzyme B Proteolytisk aktivitet: Hydrolyse av 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
Granzymes er en familie av serin-proteaser som finnes i de sekretoriske lysosomer av naturlige dreper (NK) celler og cytotoksiske T-lymfocytter (CTL) 1. Fem forskjellige granzymes eksistere hos mennesker (A, B, H, K og M), og ti i mus (A – G, K, M og N) 2,3. Granzyme A og granzyme B (GzmA, GzmB) er den mest tallrike og har blitt grundig undersøkt i den menneskelige og gnager setting.
Den klassiske funksjonen til GzmB er induksjon av apoptose i målceller som utføres i forbindelse med den pore-dannende protein perforin, som tillater granzyme å få tilgang til målcellen cytosol 4. Selv GzmB uttrykk er utvetydig funnet i cytotoksiske lymfocytter, har nyere studier er adressering en rekke andre GzmB-uttrykke celletyper, inkludert men ikke begrenset til keratinocytter 5, basofile 6, mastceller 7, plasmacytoid dendrittiske celler 8 og B-celler 9, 10.I denne sammenheng ble ikke-apoptotiske GzmB funksjoner avslørt alt fra deltakelse i inflammatoriske prosesser, vev ombygging og andre immunregulerende egenskaper 11-14.
Gitt at en bredere biologiske rolle har blitt foreslått for GzmB enn tidligere mistenkt, forskere krever pålitelige og konkrete verktøy for deteksjon sin. Av fordel er GzmB spesifikke krav for å spalte på karboksylsiden av asparaginsyrerester, bolig unike blant eukaryote serinproteaser 15. Mus, menneske og rotte GzmB er strukturelt svært like, men det utvidede substrat spesifisitet mus GzmB forskjellig diskret fra den til både human og rotte 16, noe som betyr at visse generelle substrater med Asp på terminalen (P1) kan spaltes effektivt ved GzmB fra alle tre arter, kan mens andre substrater med mer kompliserte sekvenser oppstrøms av P1 gi allment variable resultater. I både fortid og nyere literature, har dette faktum forårsaket betydelig forvirring og feiltolkning av den biologiske betydningen av noen eksperimentelle funn, selv om nøye kontrollert, har kinetiske studier søkt å rette opp situasjonen 17.
I denne artikkelen har vi forsøkt å illustrere disse punkter ved bruk av to kommersielt tilgjengelige substrater, nemlig Boc-Ala-Ala-Asp-SBzl og N-acetyl-Ile-Glu-Pro-Asp-p-nitroanilid. De to reagenser gjøre generere ulike reaktive grupper etter cleavage (en gratis sulfhydryl versus et fluorescerende gratis paranitroanilide), men dette har ingen effekt uansett på proteolysespalting. Den beskrevne protokollen er en moderne tilpasning av en svært gammel protokoll 18, men skal hjelpe etterforskerne å bruke de ulike GzmB underlag hensiktsmessig, samtidig gir en metodisk rammeverk for å oppdage aktiviteten til andre granzymes, for eksempel GzmA og GzmH.
Protocol
Representative Results
Discussion
Historisk har granzymes ble identifisert som viktige effektor molekyler av cytotoksiske lymfocytter (CTL og NK-celler) som er i stand til å indusere en hurtig apoptotisk død i målceller. Dette skyldes i hovedsak virkningen av GzmB, som kløyvde mål substratmolekyler på aspartat (D) rester og dermed var i stand til å aktivere caspase kaskade av begge spalte pro-kaspaser, samt flere av sine nedstrøms mål. Imidlertid er det nå forstås at GzmB ekspresjon ikke er begrenset til lymfoide cytotoksiske celler, og dens …
Declarações
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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