Identification of DNAJA1 as a novel interacting partner and a substrate of human transglutaminase 2.

Transglutaminase 2 (TG2) is a ubiquitously expressed multifunctional member of the transglutaminase enzyme family. It has been implicated to have roles in many physiological and pathological processes such as differentiation, apoptosis, signal transduction, adhesion and migration, wound healing and inflammation. Previous studies revealed that TG2 has various intra- and extra-cellular interacting partners, which contribute to these processes. In the present study, we identified a molecular co-chaperone, DNAJA1, as a novel interacting partner of human TG2 using a GST pull-down assay and subsequent mass spectrometry analysis, and further confirmed this interaction via ELISA and surface plasmon resonance measurements. Interaction studies were also performed with domain variants of TG2 and results suggest that the catalytic core domain of TG2 is essential for the TG2-DNAJA1 interaction. Cross-linking activity was not essential for the interaction since DNAJA1 was also found to interact with the catalytically inactive form of TG2. Furthermore, we have showed that DNAJA1 interacts with the open form of TG2 and regulates its transamidation activity under both in vitro and in situ conditions. We also found that DNAJA1 is a glutamine donor substrate of TG2. Since DNAJA1 and TG2 are reported to regulate common pathological conditions such as neurodegenerative disorders and cancer, the findings in the present paper open up possibilities to explore molecular mechanisms behind TG2-regulated functions.

Identification of a specific one amino acid change in recombinant human transglutaminase 2 that regulates its activity and calcium sensitivity.

TG2 (transglutaminase 2) is a calcium-dependent protein cross-linking enzyme which is involved in a variety of cellular processes. The threshold level of calcium needed for endogenous and recombinant TG2 activity has been controversial, the former being more sensitive to calcium than the latter. In the present study we address this question by identifying a single amino acid change from conserved valine to glycine at position 224 in recombinant TG2 compared with the endogenous sequence present in the available genomic databases. Substituting a valine residue for Gly224 in the recombinant TG2 increased its calcium-binding affinity and transamidation activity 10-fold and isopeptidase activity severalfold, explaining the inactivity of widely used recombinant TG2 at physiological calcium concentrations. ITC (isothermal titration calorimetry) measurements showed 7-fold higher calcium-binding affinities for TG2 valine residues which could be activated inside cells. The two forms had comparable substrate- and GTP-binding affinities and also bound fibronectin similarly, but coeliac antibodies had a higher affinity for TG2 valine residues. Structural analysis indicated a higher stability for TG2 valine residues and a decrease in flexibility of the calcium-binding loop resulting in improved metal-binding affinity. The results of the present study suggest that Val224 increases TG2 activity by modulating its calcium-binding affinity enabling transamidation reactions inside cells.