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

Tomasz Janiszewski, Sonia Kołt, Dion Kaiserman, Scott J. Snipas, Shuang Li, Julita Kulbacka, Jolanta Saczko, Niels Bovenschen, Guy Salvesen, Marcin Drąg, Phillip I. Bird, Paulina Kasperkiewicz

Research output: Contribution to journalArticleResearchpeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)9567-9582
Number of pages16
JournalThe Journal of Biological Chemistry
Volume295
Issue number28
DOIs
Publication statusPublished - 10 Jul 2020

Keywords

  • activity-based probe
  • amino acid
  • granzyme B
  • innate immune system
  • natural killer cells (NK cells)
  • peptide
  • protease
  • quenched fluorescence assay
  • serine protease
  • substrate specificity

Cite this

Janiszewski, T., Kołt, S., Kaiserman, D., Snipas, S. J., Li, S., Kulbacka, J., Saczko, J., Bovenschen, N., Salvesen, G., Drąg, M., Bird, P. I., & Kasperkiewicz, P. (2020). Noninvasive optical detection of granzyme B from natural killer cells with enzyme-activated fluorogenic probes. The Journal of Biological Chemistry, 295(28), 9567-9582. https://doi.org/10.1074/jbc.RA120.013204