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International audience; An extracellular form of the Ca2+-dependent protein-cross-linking enzyme transglutaminase (TGase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen transglutaminase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immuno-fluorescence staining and the in situ cross-linking of fluorescently labeled substrates. Transglutaminase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein: His6-Xpr-GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as modulator of cell wall building and strengthening. The secreted pollen TGase catalyzed the cross-linking of both polyamines (PAs) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activity was observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization.

Tissue transglutaminase (tTG) is a Ca(2+)-dependent enzyme which cross-links proteins via epsilon(gamma-glutamyl)lysine bridges. There is increasing evidence that tTG is involved in wound repair and tissue stabilization, as well as in physiological mechanisms leading to cell death. To investigate the role of this enzyme in tissue wounding leading to loss of Ca(2+) homoeostasis, we initially used a model involving electroporation to reproduce cell wounding under controlled conditions. Two cell models were used whereby tTG expression is regulated either by antisense silencing in ECV 304 cells or by using transfected Swiss 3T3 cells in which tTG expression is under the control of the tet regulatory system. Using these cells, loss of Ca(2+) homoeostasis following electroporation led to a tTG-dependent formation of highly cross-linked proteinaceous shells from intracellular proteins. Formation of these structures is dependent on elevated intracellular Ca(2+), but it is independent of intracellular proteases and is near maximal after only 20 min post-wounding. Using labelled primary amines as an indicator of tTG activity within these 'wounded cells', we demonstrate that tTG modifies a wide range of proteins that are present in both the perinuclear and intranuclear spaces. The demonstration of entrapped DNA within these shell structures, which showed limited fragmentation, provides evidence that the high degree of transglutaminase cross-linking results in the prevention of DNA release, which may serve to dampen any subsequent inflammatory response. Comparable observations were shown when monolayers of cells were mechanically wounded by scratching. In this second model of cell wounding, redistribution of tTG activity to the extracellular matrix was also demonstrated, an effect which may serve to stabilize tissues post-trauma, and thus contribute to the maintenance of tissue integrity.

Transglutaminases (Tgases) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of isopeptide bonds. This occurs either through protein cross-linking via ∊-(γ-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation, and their accumulation is found in a number of tissues and processes where such properties are important, including skin, hair, blood clotting and wound healing. However, deregulation of enzyme activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin. In the present review we detail the structural and regulatory features important in mammalian Tgases, with particular focus on the ubiquitous type 2 tissue enzyme. Physiological roles and substrates are discussed with a view to increasing and understanding the pathogenesis of the diseases associated with transglutaminases. Moreover the ability of these enzymes to modify proteins and act as biological glues has not gone unnoticed by the commercial sector. As a consequence, we have included some of the present and future biotechnological applications of this increasingly important group of enzymes.

Treatment of the hamster fibrosarcoma cell lines (Met B, D and E) and BHK-21 hamster fibroblast cells with the glucocorticoid dexamethasone led to a powerful dose-dependent mRNA-synthesis-dependent increase in transglutaminase activity, which can be correlated with dexamethasone-responsive receptor numbers in each cell line. Increasing the number of dexamethasone-responsive receptors by transfection of cells with the HG1 glucocorticoid receptor protein caused an increase in transglutaminase activity that was proportional to the level of transfected receptor. In all experiments the levels of the tissue transglutaminase-mediated detergent-insoluble bodies was found to be comparable with increases in transglutaminase activity. Despite an increase in detergent-insoluble body formation, an increase in apoptosis as measured by DNA fragmentation was not found. Incubation of cells with the non-toxic competitive transglutaminase substrate fluorescein cadaverine led to the incorporation of this fluorescent amine into cellular proteins when cells were damaged after exposure to trypsin during cell passage. These cross-linked proteins containing fluorescein cadaverine were shown to be present in the detergent-insoluble bodies, indicating that the origin of these bodies is via activation of tissue transglutaminase after cell damage by trypsinization rather than apoptosis per se,since Met B cells expressing the bcl-2 cDNA were not protected from detergent-insoluble body formation. We describe a novel mechanism of cell death related to tissue transglutaminase expression and cell damage.

Electropermeabilized human endothelial cells (ECV-304) were used to study the regulation of tissue transglutaminase (tTGase) activity in the intracellular environment. An ELSA (enzyme-linked sorbent assay) plate assay was developed for intracellular tTGase activity, using the incorporation of a biotinylated primary amine, 5-{[(N-biotinoylamino)hexanoyl]amino}pentylamine (biotin-x-cadaverine; BTC), into endogenous protein substrates of tTGase. This incorporation process was inhibited by competitive inhibitors of tTGase, cystamine and monodansylcadaverine, in a dose-dependent manner. Over a 30 min period tTGase and its protein substrates did not leak out of the cell, and no incorporation of BTC occurred in unpermeabilized cells, indicating the reaction to be intracellular. In the presence of 10 nM or 10 μM Ca2+, when nucleotides ATP and GTP were added at concentrations mimicking cytosolic levels, tTGase activity was decreased virtually to zero. Only at 100 μM Ca2+, when nucleotides were low or absent was tTGase activity observed. Under these conditions a variety of proteins was labelled by the enzyme, with the major labelling found in a protein of molecular mass around 51 kDa when analysed by SDS/PAGE/Western blotting.

Ca2+-Induced insulin release from electropermeabilised islets is inhibited by the transglutaminase inhibitors monodansylcadaverine, glycine methylester, methylamine and cystamine but not by the control compounds dimethyl monodansylcadaverine and sarcosine methylester which lack the primary amine group. Neither monodansylcadaverine nor glycine methylester inhibited insulin secretion induced by either cAMP or the phorbol ester PMA at basal levels (10 nM) of Ca2+. These data provide further evidence for the involvement of transglutaminase in Ca2+ induced insulin secretion, they also suggest that insulin secretion induced by either cAMP or PMA may act in part by a mechanism independent of that induced by Ca2+.

The sensitivity of tissue transglutaminase to activation by Ca2+ and other cellular factors was investigated using the enzyme purified from rat liver. The inclusion of Mg2+ in the assay system appeared to reduce the Ca2+-requirement of the enzyme when native N,N′-dimethylcasein was used as the protein acceptor substrate. However, when this protein was dephosphorylated, the Ca2+-requirement was unaffected by Mg2+. In addition, using this modified assay, a Km for Ca2+ was calculated to be in the range of 3–4 μM, at least an order of magnitude lower than that obtained with native acceptor substrate. Membrane phospholipids, 1,2-diolein and calmodufin were found not to affect the activation oftransglutaminase by Ca2+. The sensitivity of transglutaminase to Ca2+ which we have now demonstrated suggests that this enzyme may directly act as a receptor protein for Ca2+ during stimulusresponse coupling mediated by this cation.

Measurement of the content of polyamines in pancreatic islets indicated that no significant change in their concentration took place during glucose-stimulated insulin release. The finding, together with the absence of any effect of α-difluoromethylornithine on glucosestimulated insulin release suggested that rapid synthesis of polyamines is not involved in stimulus-secretion coupling in the β-cell. The concentration of polyamines found in islets were high enough for them to act as substrates for the Ca2+-dependent islet transglutaminase during insulin release. This was further demonstrated by the ability of islet transglutaminase to incorporate [14C]putrescine into proteins from islet homogenates and by the demonstration of an increase in the covalent incorporation of [14C]putrescine into the proteins of intact islets following their challenge with glucose. Unlike monoamine substrates of transglutaminase, putrescine failed to effectively inhibit insulin release when its intracellular concentration was increased. A role for polyamines in the secretory process through their incorporation into islet proteins is suggested.