Involvement of cell surface TG2 in the aggregation of K562 cells triggered by gluten.

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the ß-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset.

Transglutaminases in inflammation and fibrosis of the gastrointestinal tract and the liver.

Transglutaminases are a family of eight currently known calcium-dependent enzymes that catalyze the cross-linking or deamidation of proteins. They are involved in important biological processes such as wound healing, tissue repair, fibrogenesis, apoptosis, inflammation and cell-cycle control. Therefore, they play important roles in the pathomechanisms of autoimmune, inflammatory and degenerative diseases, many of which affect the gastrointestinal system. Transglutaminase 2 is prominent, since it is central to the pathogenesis of celiac disease, and modulates inflammation and fibrosis in inflammatory bowel and chronic liver diseases. This review highlights our present understanding of transglutaminase function in gastrointestinal and liver diseases and therapeutic strategies that target transglutaminase activities.

Effects of ligands on the stability of tissue transglutaminase: studies in vitro suggest possible modulation by ligands of protein turn-over in vivo.

Tissue transglutaminase catalyzes irreversible post-translational modification of specific protein substrates by either crosslinkage or incorporation of primary amines into glutamine residues, through glutamyl-amide isopeptide bonds. Modulation in vivo of these reactions (collectively called "transamidation") is brought about by both ligand dependent effects (chiefly, activation by calcium and inhibition by GTP) as well as by variation in enzyme tissue levels by transcriptional effects. Accumulating observations that the enzyme stability in vitro is greatly affected by interaction with ligands led us to postulate that also the turn-over in vivo might be modulated by ligands opening new scenarios on the regulation of the tissue transamidating activity. This proposal is consistent with data obtained in in vitro cell culture systems and has important implications for the expression of activity in vivo.

Ligand-induced conformational changes in tissue transglutaminase: Monte Carlo analysis of small-angle scattering data.

Small-angle neutron and x-ray scattering experiments have been performed on type 2 tissular transglutaminase to characterize the conformational changes that bring about Ca(2+) activation and guanosine triphosphate (GTP) inhibition. The native and a proteolyzed form of the enzyme, in the presence and in the absence of the two effectors, were considered. To describe the shape of transglutaminase in the different conformations, a Monte Carlo method for calculating small-angle neutron scattering profiles was developed by taking into account the computer-designed structure of the native transglutaminase, the results of the Guinier analysis, and the essential role played by the solvent-exposed peptide loop for the conformational changes of the protein after activation. Although the range of the neutron scattering data is rather limited, by using the Monte Carlo analysis, and because the structure of the native protein is available, the distribution of the protein conformations after ligand interaction was obtained. Calcium activation promotes a rotation of the C-terminal with respect to the N-terminal domain around the solvent-exposed peptide loop that connects the two regions. The psi angle between the longest axes of the two pairs of domains is found to be above 50 degrees, larger than the psi value of 35 degrees calculated for the native transglutaminase. On the other hand, the addition of GTP makes possible conformations characterized by psi angles lower than 34 degrees. These results are in good agreement with the proposed enzyme activity regulation: in the presence of GTP, the catalytic site is shielded by the more compact protein structure, while the conformational changes induced by Ca(2+) make the active site accessible to the substrate.

Conformational stability of human erythrocyte transglutaminase. Patterns of thermal unfolding at acid and alkaline pH.

Tissue-type transglutaminase is irreversibly inactivated during heat treatment. The rate of inactivation is low at pH 7.5; it increases slightly at acid pH (6.1) but much more at alkaline pH (9.0-9.5), suggesting that specific effects take place in the alkaline range, possibly in relation to decreased stability of the transition-state intermediate as pH is raised above 9.0. Differential scanning calorimetry experiments indicate that thermal unfolding of the protein occurs with two separate transitions, involving independent regions of the enzyme. They are assigned to domains 1 and 2 and domains 3 and 4, respectively, by a combination of calorimetric and spectroscopic techniques. When considering the effects of pH, we noted that transglutaminase was unfolded via different pathways at the different pH values considered. At acid pH, the whole structure of the protein was lost irreversibly, with massive aggregation. At neutral and, even more so, at alkaline pH, aggregation was absent (or very limited at high protein concentration) and the loss of secondary structure was dependent on the ionization state of crucial lysine residues. Unfolding at pH 9.5 apparently chiefly involved the N-terminal region, as testified by changes in protein intrinsic fluorescence. In addition, the C-terminal region was destabilized at each pH value tested during thermal unfolding, as shown by digestion with V8 proteinase, which is inactive on the native protein. Evidence was obtained that the N-terminal and C-terminal regions interact with each other in determining the structure of the native protein.

The structural basis for the regulation of tissue transglutaminase by calcium ions.

The role of calcium ions in the regulation of tissue transglutaminase is investigated by experimental approaches and computer modeling. A three-dimensional model of the transglutaminase is computed by homology building on crystallized human factor XIII and is used to interpret structural and functional results. The molecule is a prolate ellipsoid (6.2 x 4.2 x 11 nm) and comprises four domains, assembled pairwise into N-terminal and C-terminal regions. The active site is hidden in a cleft between these regions and is inaccessible to macromolecular substrates in the calcium-free form. Protein dynamics simulation indicates that these regions move apart upon addition of calcium ions, revealing the active site for catalysis. The protein dimensions are consistent with results obtained with small-angle neutron and X-ray scattering. The gyration radius of the protein (3 nm) increases in the presence of calcium ions (3.9 nm), but it is virtually unaffected in the presence of GTP, suggesting that only calcium ions can promote major structural changes in the native protein. Proteolysis of an exposed loop connecting the N-terminal and C-terminal regions is linearly correlated with enzyme inactivation and prevents the calcium-induced conformational changes.

Active site labeling of erythrocyte transglutaminase by o-phthalaldehyde.

Tissue-type transglutaminase is inactivated in a time-dependent way during incubation with submillimolar concentrations of o-phthalaldehyde, with affinity labeling kinetics. The rate of inactivation by the reagent is greatly enhanced in the presence of the essential enzyme cofactor calcium and is decreased by GTP, an allosteric inhibitor. A fluorescent isoindole derivative is formed during the modification apparently through crosslinkage of active site Cys 277 to a lysine residue. These data and the quenching of fluorescence by addition of calcium ions suggest that the enzyme active site is directly involved in the inactivation process.

Properties of diacetyl (acetoin) reductase from Bacillus stearothermophilus.

The cells of Bacillus stearothermophilus contain an NADH-dependent diacetyl (acetoin) reductase. The enzyme was easily purified to homogeneity, partially characterised, and found to be composed of two subunits with the same molecular weight. In the presence of NADH, it catalyses the stereospecific reduction of diacetyl first to (3S)-acetoin and then to (2S,3S)-butanediol; in the presence of NAD+, it catalyses the oxidation of (2S,3S)- and meso-butanediol, respectively, to (3S)-acetoin and to (3R)-acetoin, but is unable to oxidise these compounds to diacetyl. The enzyme is also able to catalyse redox reactions involving some endo-bicyclic octen- and heptenols and the related ketones, and its use is suggested also for the recycling of NAD+ and NADH in enzymatic redox reactions useful in organic syntheses.

Cardiomyocyte troponin T immunoreactivity is modified by cross-linking resulting from intracellular calcium overload.

BACKGROUND: During myocardial ischemia, the increase in cytosolic Ca2+ promotes the activation of neutral proteases such as calpains. Since the troponin T subunit is a substrate for calpains, we investigated the effects of irreversible myocyte damage on troponin T immunoreactivity. METHODS AND RESULTS: Hearts from adult guinea pigs (n=32) were perfused under conditions of normoxia, ischemia, postischemic reperfusion, or Ca2+ paradox. Hearts were frozen and processed for immunohistochemistry and Western blot with three anti-troponin T monoclonal antibodies. Two of these antibodies are unreactive on cryosections of freshly isolated and normoxic hearts and of hearts exposed to 30 minutes of no-flow ischemia. In contrast, reactivity is detected in rare myocytes after 60 minutes of ischemia, in a large population of myocytes after 60 minutes of ischemia followed by 30 minutes of reperfusion, and in every myocyte exposed to Ca2+ paradox. In Western blots, samples from ischemia-reperfusion and Ca2+ overloaded hearts show reactive polypeptides of about 240 to 260 kD and 65 to 66 kD in addition to troponin T. A similar pattern of immunoreactivity is observed with an anti-troponin I antibody. Histochemical troponin T immunoreactivity and reactivity on high-molecular-weight polypeptides are detectable in normal heart samples after preincubation with 10 mmol/L Ca2+ or with transglutaminase, whereas they are not if either transglutaminase or calpain is inhibited. CONCLUSIONS: The evolution of the ischemic injury is accompanied by changes in troponin T immunoreactivity as a consequence of the calcium-dependent activation of both calpain proteolysis and transglutaminase cross-linking.

Properties of particulate transglutaminase from Yoshida tumor cells.

Homogenates of Yoshida hepatoma cells, cultured as ascite suspension in vivo, display significant transglutaminase activity in both the cytosolic and the particulate fraction. The enzyme, however, is predominantly membrane-bound. Transglutaminase was solubilized from the membranes either by extraction with detergents or treatment with neutralized hydroxylamine or proteinases. We observed similar molecular weight under denaturing conditions, catalytic and immunologic properties for purified cytosolic and solubilized transglutaminase, and identity of the limited proteolytic maps. These results suggest that transglutaminase isoforms actually consist of the same protein undergoing translocation by unknown mechanisms.

Inactivation and cleavage of liver 6-P-gluconate dehydrogenase during irradiation in the presence of vanadate.

Lamb liver phosphogluconate dehydrogenase is inactivated and selectively cleaved during irradiation in the presence of vanadate. Under our experimental conditions, the correlation between the species of vanadate in solution and rates of enzyme inactivation and cleavage indicates tetravanadate as the most likely photosensitizing agent, in agreement with previous data on other proteins. The enzyme is inactivated more rapidly at acidic pH and is partially protected by the coenzyme NADP, but not by the substrate phosphogluconate. Complete inactivation is obtained when only half of the protein is cleaved into smaller peptides. Differences in the pattern of the peptides produced are observed when irradiation is carried out in phosphate rather than in Hepes buffer: in the former instance cleavage results into formation of a main peptide of 47 kDa, while in latter case two additional peptides of 31 and 25 kDa are produced.

Purification and properties of 6-phosphogluconate dehydrogenase from beet leaves.

We have purified to homogeneity 6-Phosphogluconate dehydrogenase from leaves of silver beet (Beta vulgaris L.) by means of cation-exchange and affinity chromatography. The enzyme is a homodimer of 52 kDa subunits; it catalyzes NADP dependent oxidation of 6-P-gluconate with Michaelian substrate saturation. The activity is affected by some intermediates of carbohydrate metabolism, particularly erythrose-4-P. Subcellular fractionation studies indicate the cytosolic location of the enzyme.

Transglutaminase-catalyzed polymerization of troponin in vitro.

In the presence of calcium ions, tissue transglutaminase catalyzes the polymerization of skeletal muscle troponin to high molecular weight insoluble aggregate. The specific action of transglutaminase is proved by the isolation of glutamyl-spermidine isopeptide derivatives. The process involves mainly the troponin T subunit (TnT), with formation of dimers and trimers of TnT, which were reactive with specific antibodies by immunoblotting. Furthermore when incubation is carried out in the presence of radioactive polyamines, the label is incorporated selectively into TnT subunits.

Structural investigation of transglutaminase by Fourier transform infrared spectroscopy.

The secondary structure of transglutaminase was investigated by Fourier transform infrared spectroscopy. Spectra of the protein in both H2O and 2H2O were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide-I band. The quantitative analysis of the amide-I-band components was made by a curve-fitting procedure. The protein was studied in the absence and in the presence of 1 mM GTP, 1 mM Ca2+ and 1 mM GTP/1 mM Ca2+. The quantitative analysis of infrared spectra revealed that no remarkable changes in the secondary structure of the enzyme are induced by GTP, Ca2+ or Ca2+/GTP. Major changes, however were observed in the thermal-denaturation behavior of the protein. The protein showed maximum of denaturation at temperatures over 50-55 degrees C in the absence or in the presence of 1 mM Ca2+ and over 55-60 degrees C in the presence of 1 mM GTP or 1 mM Ca2+/1 mM GTP. The results obtained indicate that GTP induces a stabilization of the tertiary structure of the enzyme, even in the presence of 1 mM Ca2+. The thermal denaturation patterns of the protein suggest the occurrence of Ca(2+)-dependent aggregation.

Regulation of transglutaminase activity by GTP in digitonin permeabilized Yoshida tumor cells.

Yoshida tumor cells contain consistent amounts of type 2 transglutaminase, along with a membrane bound form of the enzyme. Digitonin permeabilized cells retain a large proportion of type 2 TGase and of substrate proteins which are labelled by radioactive putrescine in the presence of calcium. GTP inhibits protein labelling at low calcium concentration by inhibiting type 2 TGase without affecting membrane-bound TGase. These results support the notion that inhibition of type 2 TGase by GTP is physiologically relevant.

Studies on tissue transglutaminases: interaction of erythrocyte type-2 transglutaminase with GTP.

Ca2+ and GTP are the main modulators of type-2 transglutaminases. To study the interaction of the enzyme with GTP, we have employed periodate-oxidized GTP as an affinity-label probe. Dialdehyde GTP bound irreversibly to type-2 transglutaminase in a time-dependent way with 1:1 stoichiometry at complete modification. The reaction took place in the absence, but was more rapid in the presence, of cyanoborohydride. Native GTP prevented incorporation of dialdehyde GTP, and Ca2+ significantly slowed down the reaction rate. The modified enzyme displayed decreased sensitivity to Ca2+, with a sigmoid saturation curve. We conclude that type-2 transglutaminase has a single GTP-binding site, the modification of which by dialdehyde GTP mimics nucleotide binding to the enzyme.

Purification of testicular transglutaminase by hydrophobic chromatography on phenyl-sepharose.

We developed a procedure for purification to homogeneity of cytosolic transglutaminase from bovine testes, by means of anion exchange, size exclusion and hydrophobic chromatography. The crucial step is chromatography on Phenyl sepharose, with specific elution by the inhibitor GTP. The enzyme displays the properties of type 2 transglutaminases.

Inactivation of placental factor XIIIa by acrylamide.

Acrylamide rapidly and irreversibly inactivates thrombin activated Factor XIIIa, without affecting neither the intact zymogen nor its proteolytic activation. The inactivation is strictly dependent on the presence of calcium ions and is accompanied by a decrease in the number of free and total thiol residues, suggesting that cysteine residue(s), whose reactivity is modulated by calcium, is (are) probably responsible for the acrylamide-directed inactivation.

Photocleavage of muscle glycogen phosphorylase by vanadate.

Glycogen phosphorylase is progressively degraded during irradiation with near UV light in the presence of vanadate. The pattern of protein cleavage by monovanadate is characterised by fewer peptides than that by decavanadate, which leads to fragmentation in a ligand dependent way. In both instances, the initial cleavage releases a peptide of 82,000 daltons which accounts for the N-terminal portion of the subunit, including the regulatory phosphorylation site.