Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters

Abstract

Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca 2+ -dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins or γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the Km and the Vmax kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2 driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of cross-linked proteins correlates with the manifestation of degenerative disorders.

Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate

“…However, fluorescence polarization was comparable both in the presence or absence of glycine methyl ester (data not shown) in concordance with our earlier observations [11] and the MS analysis has not identified transamidated peptide product in the applied assay conditions suggesting that transamidation does not have significant rate in TG2 catalysed reaction (Fig. S4) [9].…”

“…S5A and B, respectively). The increasing S100A4(GST) concentration results in dose dependent increase of isopeptidase activity of TG2 in the Zedira assay; this may be explained by the reported interaction of TG2 and S100A4 [18] leading to the potential stabilization of the active conformation of the enzyme because in previous study amine donor substrates did not influence isopeptidase activity [11]. On the other hand, in the proteinbased assay, the increasing concentration of S100A4(GST) did not lead to change of the isopeptidase activity, probably due to the relatively high ratio of S100A4(GST) concentration in the substrate which already could be in saturation without addition of extra S100A4(GST).…”

“…The Val 224 containing recombinant human TG2 (Uniprot code: P21980) and its mutants were expressed in N-terminally (His) 6 -tagged form (pET-30 Ek/LIC-TG2; Mw: 82,745 Da) and purified by Ni-NTA affinity chromatography as described previously [11].…”

“…This K m value is extremely low compared to published values for human TG2. We measured 13.3 µM and 54.3 µM K m values using Zedira assay with A101 and A102 isopeptidase substrates (deaminylation activity), respectively, in which test the substrates are peptides containing an incorporated unspecific amine (quencher labelled cadaverine) [11]. In other transglutaminase assays when deamidation was detected by kinetic spectrophotometric assay using Cbz-Gln-Gly as substrate, the K m value was in the mM range [21].…”

“…The removal of the previously incorporated monoamines (deaminylation) [8][9][10][11] and the isopeptide cleavage between short peptides [12] were demonstrated measuring fluorescence intensity change or using capillary electrophoresis. On a protein level only Factor XIIIa catalysed isopeptidase activity was detected what can reverse the incorporation of α2-plasmin inhibitor into fibrin clots potentially regulating the fibrinolytic processes [13,14].…”