Noninvasive optical detection of Granzyme B from natural killer cells using enzyme-activated fluorogenic probes

Abstract

Despite many studies on the cytotoxic protease granzyme B, key aspects of its function remain unexplored due to the lack of selective probes for its activity. In this study, we fully mapped the substrate preferences of GrB using a set of unnatural amino acids, demonstrating previously unknown GrB substrate preferences that we then used to design novel substrate-based inhibitors and a GrB-activatable activitybased probe. We showed that our GrB probes react poorly with caspases, making them ideal for the in-depth analysis of GrB localization and function in cells. With our quenched fluorescence substrate, we determined GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with the GrB attack MDA-MB-231 target cells, and active GrB influences its target cell killing efficiency.NK cell inhibitory receptors recognize MHC class I proteins and protect healthy host cells 2 . Among the various weapons of NK cells and cytotoxic T lymphocytes (CTLs), the most important, located in the cytotoxic granules, are perforin and granzymes (Granule associated enzymes, Grs). Grs are the family of homologous serine proteases and the five different human granzymes (A/B/H/K/M); the most studied and abundant are granzymes A and B 3, 4 . These enzymes are not only stored within the cytotoxic granules of immune killer cells but are also detected in primary breast carcinoma and in chondrocytes of articular cartilage 5 . Upon the activation of NK cells and binding to the target cell, the granule membrane fuses with the plasma membrane of the NK cell, and perforins (proteins that form pores in cell membranes) and granzymes are released from the cytotoxic effector cell into the intermembrane space 6, 7 . The mechanism of GrB entering the target cell through perforin-formed pores in the plasma membrane is still unclear 8 . GrB is transported into the target cell to carry out its effect. Within the cell, GrB initiates at least three distinct pathways of programmed cell death, namely, (1) the activation of caspase 3, which triggers apoptosis, (2) GrB caspase-3 substrate hydrolysis: the inhibitor of Caspase Activated DNAse (ICAD), Bid, (3) or the direct hydrolysis of lamin B. The detection of individual granzymes with antibody-related techniques is challenging due to their structural similarities; for example, GrB antibody cross-reacts with GrA and GrH 3 . For this reason, the detailed analysis of individual Grs is challenging. To overcome this obstacle, chemical approaches for developing enzymatic inhibitors and activity-based probes (ABP) as efficient tools for granzyme exploration have been attempted. However, one problem is that the substrate specificity of GrB is similar to that of some caspases (casp-8) 9 , cleaving the same substrates at the same cleavage site (synthetic: IEPD tetrapeptide 10 or natural ones: caspase-3 or BID 11 ); therefore, the reliable detection of individual Grs with either chemical or antibodyrelated techniques remains challenging 10, 12 . To date, only a few groups have...

Peptidyl Activity-Based Probes for Imaging Serine Proteases

“…After hydrolysis, the newly formed + H 3 N-peptide-fluorophore fragment bears a positive charge that reduces diffusion, as was demonstrated by Janiszewski et al (Janiszewski et al, 2020).…”

“…Recently, this strategy was improved by the incorporation of a set of unnatural amino acids that allowed for more adequate screening of enzyme catalytic preferences and better accommodation of substrates in enzyme pockets, leading to an increase in their specificity and potency (Kasperkiewicz et al, 2014). This strategy is called Hybrid Combinatorial Substrate Library (HyCoSuL), and to date, it has been used to distinguish between closely related enzymes belonging to the same family of serine proteases (NE (Kasperkiewicz et al, 2014), CatG (Kasperkiewicz et al, 2017), PR3 (Kasperkiewicz et al, 2017), NSP4 (Kasperkiewicz et al, 2017;(Kasperkiewicz et al, 2015), GrA (Kolt et al, 2020), and GrB (Janiszewski et al, 2020)) and cysteine proteases (caspases (Poreba et al, 2015; and cathepsins Poreba et al, 2018)).…”

“…One of the main issues is the possibility of the diffusion of the peptide fragment containing the fluorophore not only from the hydrolysis milieu but also from the entire cell. To circumvent this issue, different probe modifications have been proposed, such as substrate lipidation, the addition of basic amino acids, or the application of fluorophores at the C-terminal end of probes (Hu et al, 2014;Janiszewski et al, 2020). The aforementioned strengths and weaknesses of chemical reagents for serine protease imaging should be considered in probe design and will be additionally discussed in the next section.…”

“…The other type of linkers are long alkyls including commonly used 6-aminohexanoic acid (6-Ahx or Aca). This type of linker has been successfully applied in ABP for some serine proteases, including CatG, PR3, NSP4, GrA, and GrB (Kasperkiewicz et al, 2014;Kasperkiewicz et al, 2015;Kasperkiewicz et al, 2017;Janiszewski et al, 2020;Kolt et al, 2020). Despite the broad utility of 6-Ahx, this linker is hydrophobic and to compensate for the polarity it is recommended for reagents with more hydrophilic peptide part, while for more hydrophobic one, polyethers are a better choice.…”

“…Therefore, to reduce the negative influence of longer linkers, usually the peptide chain length is optimized. This type of optimization was performed for example for GrB (Janiszewski et al, 2020), where the more distant peptiderecognizing pockets, possessing broader specificity and recognizing a vast majority of amino acids, influence the kinetic parameters of enzyme-probe binding.…”