のBoc-ALA-ALA-Aspを-S-Bzlのの加水分解：具体的にグランザイムBタンパク質分解活性を測定する比色アッセイ
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
グランザイムは、ナチュラルキラー（NK）細胞および細胞傷害性Tリンパ球（CTL）1の分泌リソソームに見出されるセリンプロテアーゼのファミリーである。五つの異なるグランザイムは、ヒト（A、B、H、KとM）で存在し、そしてマウスにおける10（A – G、K、M及びN）2,3。グランザイムAおよびグランザイムB（GZMA、GZMB）が最も豊富であり、広範囲に人間と齧歯類の設定で研究されてきた。
GZMBの古典的な機能は、標的細胞の細胞質ゾル4にアクセスするためのグランザイムを可能にする孔形成タンパク質、パーフォリンと組み合わせて実行される、標的細胞におけるアポトーシスの誘導である。 GZMB発現が明確に細胞傷害性リンパ球において見出されているが、最近の研究では、好塩基球6、肥満細胞7、形質細胞様樹状細胞8、およびB細胞9を含むが、ケラチノサイト5に限定されるものではなく、他のGZMBを発現する種々の細胞型に対処されてきた、 10。この文脈では、非アポトーシスGZMB機能は炎症プロセス、組織リモデリングおよび他の免疫調節特性11-14の参加の範囲が明らかになった。
より広範な生物学的役割は、以前に疑われるよりもGZMBのために提案されていることを考えると、研究者は、その検出のための信頼性の高い、特定のツールが必要です。有利なのアスパラギン残基のカルボキシル側で切断するGZMBの特定の要件、真核生物のセリンプロテアーゼ15の中でユニークな特性である。マウス、ヒトおよびラットGZMBしかしマウスGZMBの拡張された基質特異性端子（P1）のAspを有する特定のジェネリック基質は、GZMBによって効率的に切断することができることを意味し、ヒトおよびラット16の両方とは微妙に異なるが、構造的に非常に類似しているすべての3種から、上流のP1のより複雑な配列をもつ他の基材に対し、広く多様な結果を与える可能性があります。過去と最近の李両方でterature、この事実は、注意深く制御、動態試験状況17を修正しようとしているにもかかわらず、いくつかの実験結果の生物学的意義のかなりの混乱や誤解を引き起こした。
本論文では、2つの市販の基板、すなわちのBoc-ALA-ALA-Aspを-SBzlとN-アセチルイルのGlu-Proの-ASP-p-ニトロアニリドを使用して、これらのポイントを説明しようとしてきた。 2つの試薬は、切断（蛍光無料パラニトロアニ対遊離スルフヒドリル）以下の異なる反応基を生成行いますが、これはタンパク質分解的切断には影響何もありません。記載されているプロトコルは、非常に古いプロトコル18の近代的な適応ですが、また、そのようなGZMAやGzmHなど他グランザイム、の活性を検出するための方法論的な枠組みを提供しながら、研究者は、適切に異なるGZMB基板を使用するように助けるべきである。
Protocol
Representative Results
Discussion
歴史的にグランザイムは、標的細胞の急速なアポトーシス死を誘導することができる細胞傷害性リンパ球（CTLおよびNK細胞）の重要なエフェクター分子として同定された。これは主に、アスパラギン酸（D）残基で標的基質分子を切断し、したがって両方の切断プロカスパーゼによってカスパーゼカスケードを活性化することができるだけでなく、それらの下流の標的のいくつかあったGZMBの作…
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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