Effects of ligands on structure and stability of Tissue Trasglutaminase: a calorimetric and spectroscopic study

Transglutaminases (Tgases) are a wide family of enzymes whose main role is to catalyze calcium dependent acyl-transfer reactions from peptidyl glutamine residues to accepting primary amines. The amine substrates can be provided by either a soluble amine (usually polyamines or alternatively histamine) and/or the aminogroups of a protein-bound lysine residue, resulting into release of proteins which are modified at glutamine residues either by simple covalent incorporation of amines or by crosslinkage through proteinase-resistant isopeptide bonds.In mammals, eight distinct Tgase isoenzymes have been identified at the genomic level. The most extensively investigated isoenzyme is the ubiquitous cytosolic isoform, denoted tissue transglutaminase. Its physiologic function is still uncertain and it has been proposed to be involved into as different cell reactions as programmed cell death death and stimulation to proliferation. Tissue transglutaminase is a bifunctional enzyme displaying also GTP hydrolysis along with the transamidating activity. These activities are switched on-off alternatively. Transamidation is usually a latent activity, thanks to a mechanism involving conformational changes triggered by calcium (an activator essential cofactor in transamidation) and GTP itself (an inhibitor). The aim of this study is to investigate the stability of the enzyme and the effects of ligands thereupon, employing heat as a simple quantificable destabilizing stimulus.In these studies we employed Tgase purified to homogeneity from human erythrocytes. In thermal inactivation experiments, Tgase was submitted to heat treatment at various temperature, measuring residual activity at timed intervals. Along with effects on activity, we investigated structural perturbations of the protein by Differential Scanning Calorimetry (DSC), with heating cycles employing thermal gradient of 0.8°C/min in a temperature range between 20°C to 90°C to study the unfolding process in the absence and in the presence of ligands. Finally, selective perturbations of domains 1 and 2 of the enzyme were monitored by tryptophan fluorescence emission between 310 and 370 nm, with excitation at 295 nm, at constant temperature increments. The calculated ratio of fluorescence emission at two wavelengths (at 350 and 330 nm, marked F350/F330) was a suitable measure of the loss of protein tertiary structure and was employed to measure ligand-induced conformational changes and their relationships to thermal stability. The regulation of tissue transglutaminase is achieved through conformational changes triggered by the modulatory ligands calcium (an essential activator) and GTP (an inhibitor), through induction of conformational changes. In this report we address the thermodynamics of these effects by means of a combination of thermal treatment, spectroscopic measurements and differential scanning calorimetry. Our data confirm that binding of ligands induce large conformational changes through movement of domains, identified by distinct unfolding transitions in DSC thermograms. In particular they demonstrate that the protein structure is characterized by tight interaction between the N-terminal and the C-terminal domains in the absence of ligands, a reinforcement of the interaction in the presence of the inhibitor GTP and a losening in the presence of calcium ions, with appreciable changes in thermal stability, hindering or disclosing the active site to the substrate. Also addition of heparin did not by itself significantly modify the thermogram profile but it prevented completely the aggregation of the protein which is usually observed late in the fusion profile after the unfolding process is completed.