Noninvasive optical detection of granzyme B from natural killer cells with enzyme-activated fluorogenic probes

• Published in: The Journal of biological chemistry Vol. 295; no. 28; pp. 9567 - 9582
• Main Authors: Janiszewski, Tomasz, Kołt, Sonia, Kaiserman, Dion, Snipas, Scott J., Li, Shuang, Kulbacka, Julita, Saczko, Jolanta, Bovenschen, Niels, Salvesen, Guy, Drąg, Marcin, Bird, Phillip I., Kasperkiewicz, Paulina
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.07.2020; American Society for Biochemistry and Molecular Biology
• Subjects: activity-based probe; amino acid; Cell Line, Tumor; Fluorescent Dyes - chemistry; granzyme B; Granzymes - chemistry; Granzymes - metabolism; Humans; innate immune system; Methods and Resources; natural killer cells (NK cells); Optical Imaging; peptide; Peptides - chemistry; protease; quenched fluorescence assay; serine protease; substrate specificity; T-Lymphocytes - enzymology; protease; granzyme B; peptide; quenched fluorescence assay; amino acid; substrate specificity; activity-based probe; natural killer cells (NK cells); serine protease; innate immune system
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA120.013204

Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate.