Detection of Active Granzyme A in NK92 Cells with Fluorescent Activity-Based Probe

Sonia Kołt,Guy Salvesen,Paulina Kasperkiewicz,Marcin Dra̧G,Phillip I Bird,Tomasz Janiszewski,Sylwia Modrzycka,Dion Kaiserman,Scott J Snipas

doi:10.1021/acs.jmedchem.9b02042

Abstract

Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.

Highlights

Granule-associated serine proteases (Grs) play a pivotal role in the immune system and are released by cytotoxic natural killer cells (NKs) and cytotoxic T-lymphocytes (CTLs) to eliminate abnormal cells, such as those infected with bacteria or viruses, or cancer cells.[1,2] Grs are stored in granules within the killer cell which, on engagement with a target cell, fuse with the killer cell membrane releasing their contents into the synaptic space
We tested the activity of human Granzyme A (GrA) against individual P1 library components, and our results were in accordance with existing data concerning natural amino acids
We found that GrA interacts almost exclusively with positively charged amino acid residues, with the highest activity toward ones that contain a guanidine group (L-Arg, 92% and L-Phe(guan), 100%)

Summary

Introduction

Granule-associated serine proteases (Grs) play a pivotal role in the immune system and are released by cytotoxic natural killer cells (NKs) and cytotoxic T-lymphocytes (CTLs) to eliminate abnormal cells, such as those infected with bacteria or viruses, or cancer cells.[1,2] Grs are stored in granules within the killer cell which, on engagement with a target cell, fuse with the killer cell membrane releasing their contents into the synaptic space. Besides Grs, granules contain the pore forming cytolytic protein (perforin). Granzyme A (GrA) is the only Gr that forms a dimer and is located at the same branch of an evolutionary tree as Granzyme K (GrK), found in the tryptase cluster at chromosome 5. These enzymes share 39.7% similarity (with 89% certainty), representing one of the most complementary pairs of Grs. These enzymes share 39.7% similarity (with 89% certainty), representing one of the most complementary pairs of Grs Both enzymes are trypsin-like proteases and cleave after basic amino acid residues. GrA and GrB share some substrates, and among them, 22 were cleaved in the same region but not at the same site,[5] while GrB antibodies were found to label GrA, providing strong evidence of their similarity.[6]

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Conclusion