Global identification and analysis of isozyme-specific possible substrates crosslinked by transglutaminases using substrate peptides in mouse liver fibrosis

Abstract

The transglutaminase (TG) family comprises eight isozymes that form the isopeptide bonds between glutamine and lysine residues and contribute to the fibrotic diseases via crosslinking-mediated stabilization of ECM and the activation of TGF-β in several tissues. However, despite a growing body of evidence implicating TG2 as a key enzyme in fibrosis, the causative role of TG2 and the involvement of the other isozymes have not yet been fully elucidated. Therefore, here we clarified the distributions of TG isozymes and their in situ activities and identified the isozyme-specific possible substrates for both TG1 and TG2 using their substrate peptides in mouse fibrotic liver. We found that TG1 activity was markedly enhanced intracellularly over a widespread area, whereas TG2 activity increased in the extracellular space. In total, 43 and 42 possible substrates were identified for TG1 and TG2, respectively, as involved in chromatin organization and cellular component morphogenesis. These included keratin 18, a biomarker for hepatic injury, which was accumulated in the fibrotic liver and showed the partly similar distribution with TG1 activity. These findings suggest that TG1 activity may be involved in the functional modification of intracellular proteins, whereas TG2 activity contributes to the stabilization of extracellular proteins during liver fibrosis.

Genetic variants in COL13A1, ADIPOQ and SAMM50 , in addition to the PNPLA3 gene, confer susceptibility to elevated transaminase levels in an admixed Mexican population

“…Transglutaminases (TGs) are Ca(2+)-dependent enzymes that catalyze the formation of covalent bonds between glutamine and lysine residues, and contribute to fibrotic diseases via crosslinking-mediated stabilization of extracellular matrix (Eckert et al, 2014; Iismaa et al, 2009; Lorand and Graham, 2003). TGM1 , TGM3 and TGM5 have been associated with mouse liver fibrosis and altered healing processes (De Koning et al, 2012; Tatsukawa et al, 2017), which is interesting since NAFLD can progress to cirrhosis. According to the tissue-specific pattern of mRNA expression profiles from 79 human tissues and cell types (http://biogps.org), TGM5 is highly expressed in the liver, although the precise role of TGM5 in NAFLD is not known.…”

Transglutaminase diseases: from biochemistry to the bedside

“…TG was linked with liver fibrosis in 2001 (73) using an e-(g-glutamyl) Lys crosslink antibody that has since been reported to be nonspecific (74). TG isozyme-specific substrates have demonstrated an increase in the activities of intracellular TG1 and of extracellular TG2 during liver fibrosis (75), implicating TG2 in ECM crosslinking. Effective reduction of liver fibrosis has been reported using competitive inhibitors of TG (76), such as cystamine (a nonspecific enzyme inhibitor that interferes with sulfhydryl enzymes), and without using a proper (e.g., tertiary ammonium) control.…”

“…Effective reduction of liver fibrosis has been reported using competitive inhibitors of TG (76), such as cystamine (a nonspecific enzyme inhibitor that interferes with sulfhydryl enzymes), and without using a proper (e.g., tertiary ammonium) control. However, 2 independent groups have shown that TG2 knockout mice are not altered in collagen crosslinking or liver fibrosis (75). Again, a comparison of TG isozyme-specific activities before and after induction of liver fibrosis in TG2 knockout mice has not been reported, so it is not clear if there are disease-related compensatory changes in TG isozymes in the ECM in response to TG2 deletion or if liver fibrosis is TG independent.…”

Studies on differentiation‐dependent expression and activity of distinct transglutaminases by specific substrate peptides using three‐dimensional reconstituted epidermis

“…These peptides have been widely used as tools to efficiently visualize enzyme activity in several tissues including skin epidermis . Moreover, possible substrates have been identified in feasible and efficient procedures .…”