Search

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.

Transglutaminases (TGs) catalyze the covalent crosslinking of proteins via isopeptide bonds. The most prominent isoform, TG2, is associated with physiological processes such as extracellular matrix (ECM) stabilization and plays a crucial role in the pathogenesis of e.g., fibrotic diseases, cancer and celiac disease. Therefore, TG2 represents a pharmacological target of increasing relevance. The glycosaminoglycans (GAG) heparin (HE) and heparan sulfate (HS) constitute high-affinity interaction partners of TG2 in the ECM. Chemically modified GAG are promising molecules for pharmacological applications as their composition and chemical functionalization may be used to tackle the function of ECM molecular systems, which has been recently described for hyaluronan (HA) and chondroitin sulfate (CS). Herein, we investigate the recognition of GAG derivatives by TG2 using an enzyme-crosslinking activity assay in combination with in silico molecular modeling and docking techniques. The study reveals that GAG represent potent inhibitors of TG2 crosslinking activity and offers atom-detailed mechanistic insights.

The transamidase activity of transglutaminase 2 (TGase 2) is considered to be important for several pathophysiological processes including fibrotic and neoplastic tissue growth, whereas in healthy cells this enzymatic function is predominantly latent. Methods that enable the highly sensitive detection of TGase 2, such as application of radiolabeled activity-based probes, will support the exploration of the enzyme's function in various diseases. In this context, the radiosynthesis and detailed

A reliable solution-phase synthesis of the water-soluble dipeptidic fluorogenic transglutaminase substrate Z-Glu(HMC)-Gly-OH is presented. The route started from Z-Glu-OH, which was converted into the corresponding cyclic anhydride. This building block was transformed into the regioisomeric α- and γ-dipeptides. The key step was the esterification of Z-Glu-Gly-OtBu with 4-methylumbelliferone. The final substrate compound was obtained in an acceptable yield and excellent purity without the need of purification by RP-HPLC. The advantage of this acyl donor substrate for the kinetic characterisation of inhibitors and amine-type acyl acceptor substrates is demonstrated by evaluating commercially available or literature-known irreversible inhibitors and the biogenic amines serotonin, histamine and dopamine, respectively.

Tissue transglutaminase (TGase 2) is the most abundantly expressed enzyme of the transglutaminase family and involved in a large variety of pathological processes, such as neurodegenerative diseases, disorders related to autoimmunity and inflammation as well as tumor growth, progression and metastasis. As a result, TGase 2 represents an attractive target for drug discovery and development, which requires assays that allow for the characterization of modulating agents and are appropriate for high-throughput screening. Herein, we report a fluorescence anisotropy-based approach for the determination of TGase 2's transamidase activity, following the time-dependent increase in fluorescence anisotropy due to the enzyme-catalyzed incorporation of fluorescein- and rhodamine B-conjugated cadaverines 1-3 (acyl acceptor substrates) into N,N-dimethylated casein (acyl donor substrate). These cadaverine derivatives 1-3 were obtained by solid-phase synthesis. To allow efficient conjugation of the rhodamine B moiety, different linkers providing secondary amine functions, such as sarcosyl and isonipecotyl, were introduced between the cadaverine and xanthenyl entities in compounds 2 and 3, respectively, with acyl acceptor 3 showing the most optimal substrate properties of the compounds investigated. The assay was validated for the search of both irreversible and reversible TGase 2 inhibitors using the inactivators iodoacetamide and a recently published L-lysine-derived acrylamide and the allosteric binder GTP, respectively. In addition, the fluorescence anisotropy-based method was proven to be suitable for high-throughput screening (Z' factor of 0.86) and represents a non-radioactive and highly sensitive assay for determining the active TGase 2 concentration.

Transglutaminase 2 (TGase 2)-catalyzed transamidation represents an important post-translational mechanism for protein modification with implications in physiological and pathophysiological conditions, including fibrotic and neoplastic processes. Consequently, this enzyme is considered a promising target for the diagnosis of and therapy for these diseases. In this study, we report on the synthesis and kinetic characterization of N

Small glutamate-containing peptides bearing coumarin derivatives as fluorescent leaving groups attached to the γ-carboxylic acid group of the Glu residue were synthesised and investigated with regard to their potential to act as substrates for transglutaminase 2 (TGase 2). Their synthesis was accomplished by an efficient solid-phase approach. The excellent water solubility of the compounds enabled their extensive kinetic characterisation in the context of TGase 2-catalysed hydrolysis and aminolysis. The influence of the coumarin skeleton's substitution pattern on the kinetic properties was studied. Derivatives containing 7-hydroxy-4-methylcoumarin (HMC) revealed properties superior to those of their 7-hydroxycoumarin counterparts; analogous amides are not accepted as substrates. Z-Glu(HMC)-Gly-OH, which exhibited the best substrate properties out of the investigated derivatives, was selected for representative kinetic characterisation of acyl acceptor substrates and irreversible inhibitors.

Tissue transglutaminase (transglutaminase 2) is a multifunctional enzyme with many interesting properties resulting in versatile roles in both physiology and pathophysiology. Herein, the particular involvement of the enzyme in human diseases will be outlined with special emphasis on its role in cancer and in tissue interactions with biomaterials. Despite recent progress in unraveling the different cellular functions of transglutaminase 2, several questions remain. Transglutaminase 2 features in both confirmed and some still ambiguous roles within pathological conditions, raising interest in developing inhibitors and imaging probes which target this enzyme. One important prerequisite for identifying and characterizing such molecular tools are reliable assay methods to measure the enzymatic activity. This digest Letter will provide clarification about the various assay methods described to date, accompanied by a discussion of recent progress in the development of inhibitors and imaging probes targeting transglutaminase 2.