Application of a Fluorescence Anisotropy-Based Assay to Quantify Transglutaminase 2 Activity in Cell Lysates

Abstract

Transglutaminase 2 (TGase 2) is a multifunctional protein which is involved in various physiological and pathophysiological processes. The latter also include its participation in the development and progression of malignant neoplasms, which are often accompanied by increased protein synthesis. In addition to the elucidation of the molecular functions of TGase 2 in tumor cells, knowledge of its concentration that is available for targeting by theranostic agents is a valuable information. Herein, we describe the application of a recently developed fluorescence anisotropy (FA)-based assay for the quantitative expression profiling of TGase 2 by means of transamidase-active enzyme in cell lysates. This assay is based on the incorporation of rhodamine B-isonipecotyl-cadaverine (R-I-Cad) into N,N-dimethylated casein (DMC), which results in an increase in the FA signal over time. It was shown that this reaction is not only catalyzed by TGase 2 but also by TGases 1, 3, and 6 and factor XIIIa using recombinant proteins. Therefore, control measurements in the presence of a selective irreversible TGase 2 inhibitor were mandatory to ascertain the specific contribution of TGase 2 to the overall FA rate. To validate the assay regarding the quality of quantification, spike/recovery and linearity of dilution experiments were performed. A total of 25 cancer and 5 noncancer cell lines were characterized with this assay method in terms of their activatable TGase 2 concentration (fmol/µg protein lysate) and the results were compared to protein synthesis data obtained by Western blotting. Moreover, complementary protein quantification methods using a biotinylated irreversible TGase 2 inhibitor as an activity-based probe and a commercially available ELISA were applied to selected cell lines to further validate the results obtained by the FA-based assay. Overall, the present study demonstrates that the FA-based assay using the substrate pair R-I-Cad and DMC represents a facile, homogenous and continuous method for quantifying TGase 2 activity in cell lysates.

Preclinical evaluation of an 18F-labeled Nε-acryloyllysine piperazide for covalent targeting of transglutaminase 2

“…A375, A375-hS100A4, NCI-H292, and MDA-MB-231, as well as one TGase 2-negative cell lines, i.e. MeWo, were selected based on recently published results from WesternBlot analysis and TGase 2 activity measurements in cell lysates (Hauser et al 2022 ). Cell uptake was also studied in the presence of 10 µM 7b or 100 µM verapamil.…”

Cell-Impermeable Inhibitors Confirm That Intracellular Human Transglutaminase 2 Is Responsible for the Transglutaminase-Associated Cancer Phenotype

“…While this manuscript was in preparation, Hauser et al published a novel biotinylated TG2 inhibitor ( Figure S12 ) that is highly efficient, exhibiting a k inact / K I value of 7260 M −1 s −1 (or 435,600 M −1 min −1 ) [ 53 ]. It is noteworthy that this inhibitor exploits the same binding pocket as our inhibitors disclosed herein, implying that this site of similar inhibitor scaffolds is broadly tailorable, with minimal impact on binding affinity.…”