Detection of substrates of keratinocyte transglutaminase in vitro and in vivo using a monoclonal antibody to dansylcadaverine.

A method providing more sensitive detection of transglutaminase substrates was developed to localize transglutaminase activity in tissue and to identify in vivo substrates in epidermal extracts. The enhanced sensitivity of this method was achieved via the generation of a monoclonal antibody (designated E7) made to dansylcadaverine. Transglutaminase substrates were visualized by western blot after a 1-min incubation with dansylcadaverine in contrast to the 2 h required when [14C]putrescine incorporation was measured by autoradiography of SDS-polyacrylamide gels. In addition, putative substrates not apparent using conventional methods were readily detected by western analysis. An ELISA assay to measure transglutaminase activity showed similar sensitivity to the traditional radiometric assay (Lorand et al., 1972). The correlation between the ELISA procedure and the radiometric assay was high (r2 = 0.924). Strips of neonatal human and mouse skin incubated in dansylcadaverine-supplemented culture medium were used to localize enzyme activity and to detect substrates in vivo. Transglutaminase activity was demonstrated at the cellular periphery in the upper spinous and granular cell layers of the epidermis. Substrates detected in epidermal extracts were similar to those detected using the in vitro assay. This technique allows for highly sensitive and nonradiometric analysis of both enzymatic activity and the substrates involved. The extension of this methodology to an in vivo system is the first demonstration of a system in which the dynamics of cornified envelope assembly may be further studied.     

Human mononuclear leukocyte transglutaminase activity is enhanced by streptococcal erythrogenic toxin and a staphylococcal mitogenic factor associated with toxic shock syndrome

Transglutaminase activity in human peripheral lymphocytes is enhanced after incubation of the cells with concanavalin A. Streptococcal proliferative factor toxin (erythrogenic toxin) from Streptococcus pyogenes and Toxic shock syndrome toxin from Staphylococcus aureus were purified and tested for their ability to enhance transglutaminase activity. Mononuclear leukocyte transglutaminase activity was enhanced 3–5-fold 30 min after incubation with either toxin. Enhancement occurred only when toxin was incubated with intact cells; addition of toxin to cell lysates was without effect. Transglutaminase was not measurable extracellularly. Histamine and dansyl cadaverine, competitive substrates for transglutaminase, inhibited [³H]putrescine incoporation into casein and [³H]thymidine incorporation into DNA. Incubation of lymphocytes with cycloheximide and either toxin or concanavalin A did not inhibit enzyme activity. These bacterial toxins, like phytomitogens, may perturb the cellular membrane and mediate their effect by transglutaminase-mediated cross-linking of membrane proteins.     

Dissociation of fibrinogen and fibronectin binding from transglutaminase-mediated cross-linking at the hepatocyte surface

The interaction of fibrinogen and fibronectin with hepatocytes has been dissociated into distinct binding and cross-linking steps. Binding and cross-linking of 125I-labeled ligands were both decreased by transglutaminase inhibitors, but not by heparin or hirudin. Transglutaminase activity was manifest by Ca2+-dependent incorporation of [14C]putrescine into cells. Preferential cross-linking of fibrinogen A alpha over gamma chains, and lack of inhibition by heparin or hirudin indicates the involvement of tissue transglutaminase, and not Factor XIIIa. Hepatic transglutaminase activity, as well as binding and cross-linking of fibrinogen and fibronectin, were maximally supported by Ca2+, partially supported by Mn2+ and Sr2+, and markedly decreased by Mg2+ and Ba2+. In contrast, Co2+ supported binding but not cross-linking or transglutaminase activity, indicating that binding and cross-linking are dissociable events. This conclusion was corroborated by the finding that fibrinogen fragments D95 and D78 both inhibited Ca2+-dependent fibrinogen binding without being cross-linked themselves. Ligand binding in the presence of either cation was localized to the cell surface as evidenced by its trypsin sensitivity. Thus, fibrinogen and fibronectin binding to hepatocytes is independent of transglutaminase activity, whereas cross-linking of these adhesive macromolecules requires an enzymatically active cellular transglutaminase. In addition, fibrinogen binding appears to be mediated by molecular determinants present in fragment D78.     

Correlation of changes in transglutaminase activity and polyamine content of neoplastic tissue during the metastatic process

Transglutaminase activity and the levels of the polyamines putrescine, spermidine and spermine were measured in two transplantable rat sarcomata: P8 which metastasises consistently to the lung, and P7 which metastasises infrequently. With the P7 sarcoma no metastases were detected following implantation; similarly, no significant changes occurred in the levels of transglutaminase activity, putrescine, spermidine or spermine during tumour growth. However, with the P8 sarcoma at approx. 30 days after implantation there was a marked decrease in transglutaminase activity, mirrored exactly by a 20-fold increase in the levels of acid-soluble putrescine. Measurement of covalently-bound polyamines in the P8 sarcoma indicated a significant and corresponding decrease in the levels of bound putrescine. The timing of these changes coincided with the time at which the P8 sarcoma was shown to have metastasised, and suggests that the changes observed may be related to this phenomenon.     

The subcellular localization of transglutaminase in normal liver and in glucagon-treated and partial hepatectomized rats

Rat liver was homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density-gradient centrifugation. Transglutaminase, when assayed with putrescine and dimethylcasein as substrates, showed three distinct localizations, cytosol (73%), plasma membrane (20%), and nuclei (7%). The distribution was unaffected by homogenization in the presence of potassium chloride, indicating that the particulate activity was not due to adsorbed cytosolic enzyme. The specific activity and subcellular distribution of transglutaminase in rats which had received intra-peritoneal glucagon, stimulating endocytosis, or which had been subjected to sub-total hepatectomy 2, 16, or 32 h previously, showed no significant difference from control animals.     

Expression of the cytosolic and particulate forms of transglutaminase during chemically induced rat liver carcinogenesis

Transglutaminase (EC 2.3.2.13) activity in chemically induced rat hepatocellular carcinomas was reduced by some 65% when compared to normal rat livers. The majority of the remaining activity (approx. 85%) was found in the particulate fraction. The use of non-ionic detergent to extract the transglutaminase activity present in both normal and tumour tissue followed by its separation on a Mono-Q column revealed two distinct peaks of activity. These peaks of activity were equivalent to those previously identified as a membrane-bound transglutaminase and the more characteristic cytosolic or tissue transglutaminase. The ratio of the activity of the cytosolic enzyme to that of the membrane-bound enzyme in normal liver was calculated as 5:1. In hepatocellular carcinomas, this ratio was reduced to 0.4:1. No significant change in the activity of the membrane-bound enzyme was detectable in tumour tissue. Comparison of the cytosolic enzyme found in hepatocellular carcinomas with that found in normal liver indicated no change in its molecular weight, Km,app for putrescine incorporation into N,N'-dimethylcasein and sensitivity to activation by Ca2+. These observations suggest that the reduction in transglutaminase activity observed in the hepatocellular carcinoma is due to a selective reduction in the expression of the cytosolic transglutaminase.     

Correlation of changes in transglutaminase activity and polyamine content of neoplastic tissue during the metastatic process

Transglutaminase activity and the levels of the polyamines putrescine, spermidine and spermine were measured in two transplantable rat sarcomata: P₈ which metastasises consistently to the lung, and P₇ which metastasises infrequently. With the P₇ sarcoma no metastases were detected following implantation; similarly, no significant changes occurred in the levels of transglutaminase activity, putrescine, spermidine or spermine during tumour growth. However, with the P₈ sarcoma at approx. 30 days after implantation there was a marked decrease in transglutaminase activity, mirrored exactly by a 20-fold increase in the levels of acid-soluble putrescine. Measurement of covalently-bound polyamines in the P₈ sarcoma indicated a significant and corresponding decrease in the levels of bound putrescine. The timing of these changes coincided with the time at which the P₈ sarcoma was shown to have metastasised, and suggests that the changes observed may be related to this phenomenon.     

Transglutaminase and neuronal differentiation

Summary During mouse brain maturation cellular transglutaminase specific activity increases 2.5 fold from day 3 to adulthood. A more pronounced increase is seen during morphological differentiation of mouse neuroblastoma cells, where serum withdrawal induces neurite outgrowth concomitant with a 10 fold increase in transglutaminase specific activity. In contrast, non-dividing neuroblastoma cells lacking neurites show only a 1.5 fold increase in enzyme specific activity. Transglutaminase activity does not reach maximal levels until extensive neurite formation has occurred. More than 80% of the transglutaminase activity is found in the soluble component of brain and neuroblastoma homogenates. Using [³H]-putrescine as the acyl acceptor, endogenous acyl donor substrates in the neuroblastoma cells included proteins that comigrated on SDS-PAGE with tubulin and actin; however, very high molecular weight crosslinked material is the major reaction product in vitro. When purified brain tubulin, microtubule associated proteins and microtubules were compared as exogenous substrates, only the polymeric microtubules were a good acyl donor substrate. Furthermore, preincubation of purified tubulin with transglutaminase and putrescine stimulated both the rate and extent of microtubule assembly. These findings suggest that transglutaminase may mediate covalent cross-linking of microtubules to other cellular components, or the post-translational modification of tubulin by the formation of -glutamylamines.     

Cellular transglutaminase. The particulate-associated transglutaminase from chondrosarcoma and liver: partial purification and characterization

A transglutaminase from the malignant chondrocytes, rat swarm chondrosarcoma cells, was partially purified and characterized in an effort to understand transformation-induced changes in its activity. This enzyme separated by DE52 column chromatography after extraction from the particulate fraction of cell lysate was found to be distinct from previously characterized transglutaminases in its electrophoretic mobility, molecular size, substrate specificity, and immunologic reactivity. This enzyme was identified as a transglutaminase by its catalysis of amine (putrescine, spermine) incorporation at the carboxamide group of protein-bound gamma-glutamyl residues, and accordance of its kinetic data with the modified double displacement mechanism described for other transglutaminases. Limited proteolysis of the isolated enzyme resulted in a 3-4-fold increase of catalytic activity and a concomitant reduction of molecular size by approximately one-half. Incubation of labeled amine with chondrosarcoma cell lysate resulted in labeling of only a few proteins that appeared to be extensively cross-linked and that were located mostly in the particulate fraction of the cells. Transglutaminase extracted from the rat liver particulate fraction displayed enzymatic and structural properties closely resembling those of the enzyme from chondrosarcoma cells.     

Free and protein-conjugated polyamines in mouse epidermal cells. Effect of high calcium and retinoic acid

We have investigated polyamine metabolism in primary cultures of mouse epidermal cells. These cells, which grow at low Ca2+ levels as a monolayer with characteristics of basal cells, terminally differentiate when the extracellular Ca2+ level is raised above 1 mM. The cellular levels of free polyamines were measured, and, after incubation of cell cultures with [3H]putrescine, the distribution of label in both acid-soluble and acid-insoluble cellular components was examined. Free polyamine levels were reduced in cells induced to differentiate. Treatment with retinoic acid, which prevents differentiation and causes increased proliferation, resulted in an increase in free putrescine. Upon adjustment of the calcium concentration to a level that induces differentiation, the enzyme transglutaminase was activated, and a concomitant increase in the level of both protein-bound mono- and bis-gamma-glutamyl derivatives of putrescine and spermidine was observed. Isolation of a material of apparent molecular weight about 6000 which contains only mono-gamma-glutamylpolyamines and the finding of both mono- and bis-gamma-glutamylpolyamines in the protein fraction containing cornified cell envelopes provided the basis for speculation on polyamines in envelope formation. Our data suggest that polyamines play a role during epidermal cell differentiation through transglutaminase-mediated post-translational modification.     

Ca-dependent inactivation of acetylcholine receptors by an endogenous transglutaminase

The nicotinic acetylcholine receptor (nAChR) from Torpedo californica and T. marmorata electric tissue polymerises irreversibly when DTE and Ca²⁺ are added to receptor-rich membranes. The polymerisation is time-dependent and complete within 3 h at 30°C. It can be completely prevented by EGTA or the transglutaminase inhibitor cystamine. Transglutaminase activity can also be monitored with the exogenous substrates [³H]putrescine and dimethylcasein. This assay can also be inhibited by EGTA or cystamine.     

Bovine aortic endothelial cell transglutaminase. Enzyme characterization and regulation of activity.

Bovine aortic endothelial cells contain Ca2+-dependent tissue-type transglutaminase. Its activity in these cells was high, with apparent Km and Vmax. values with respect to putrescine of 0.203 mM and 18.5 nmol/min per mg of protein, and its activity was inhibited by the three competitive inhibitors dansylcadaverine, spermine and methylamine. The molecular mass of endothelial cell transglutaminase estimated by gel filtration chromatography was 88 kDa and it was immunoprecipitated by rabbit monospecific antiserum raised against rat liver transglutaminase. Its enzymic activity rose when the cell cultures reached confluence, and was further increased when their proliferation was arrested (synchronized at G0/G1 phase). Most of the enzymic activity was found in the 15,000 g soluble fraction, with only 4-22% of the activity found in the particulate fraction, depending on the state of cell proliferation. Examination of these cellular fractions by SDS/polyacrylamide-gel electrophoresis and immunoblotting revealed that at confluence endothelial cells have accumulated transglutaminase antigen in their 15,000 g particulate fraction. A series of experiments demonstrated the existence of a latent transglutaminase form in non-proliferating cells, and suggested that this might involve the formation of an inhibitory complex. Treatment of cell lysates and the 15,000 g particulate fraction with high salt concentration showed a significant increase in transglutaminase activity. Mixing experiments using the 100,000 g particulate fraction or purified rat liver transglutaminase on one hand and the cytosolic fraction on the other showed dose-dependent inhibition of the transglutaminase activity of the latter. It is concluded that endothelial cells contain a particulate fraction-residing inhibitor of transglutaminase which interacts via ionic interaction with the enzyme.     

Hierarchies of protein cross-linking in the extracellular matrix: involvement of an egg surface transglutaminase in early stages of fertilization envelope assembly

The involvement of transglutaminase activity in fertilization envelope (FE) formation was investigated using eggs from the sea urchin, Strongylocentrotus purpuratus. Eggs fertilized in the presence of the transglutaminase inhibitors, putrescine and cadaverine, had disorganized and expanded FEs with inhibition of the characteristic I-T transition. The permeability of the FE was increased by these agents, as revealed by the loss of proteins from the perivitelline space and the appearance of ovoperoxidase activity in supernates from putrescine- treated eggs. [3H]putrescine was incorporated into the FE during fertilization in a reaction catalyzed by an egg surface transglutaminase that could also use dimethylcasein as a substrate in vitelline layer-denuded eggs. Egg secretory products alone had no transglutaminase activity. The cell surface transglutaminase activity was transient and maximal within 4 min of activation. The enzyme was Ca2+ dependent and was inhibited by Zn2+. We conclude that sea urchin egg surface transglutaminase catalyzes an early step in a hierarchy of cross-linking events during FE assembly, one that occurs before ovoperoxidase-mediated dityrosine formation (Foerder, C. A., and B. M. Shapiro. 1977. Proc. Natl. Acad. Sci. USA. 74:4214-4218). Thus it provides a graphic example of the physiological function of a cell surface transglutaminase.     

Changes in polyamine content, arginine and ornithine decarboxylases and transglutaminase activities during light/dark phases (of initial differentiation) in maize calluses and their chloroplasts

Maize calluses and their isolated chloroplasts were analysed to study the changes in polyamine content, arginine and ornithine decarboxylases and transglutaminase activities during light/dark phases of the first day after subculture in maintenance medium (containing 2,4-D) and differentiation medium (without 2,4-D). Free polyamine content changed significantly in both differentiating calluses and chloroplasts showing a maximum during light phase and also increasing after mid-dark phase. Acid-insoluble polyamines showed a similar trend. In whole cells from the callus cultured in maintenance medium, the changes were not significant, except for free putrescine which increased in the dark phase. In chloroplasts of both types of calluses, the trend was similar. Arginine decarboxylase activity in vitro assayed in optimal conditions was not affected by hormone deprivation either in whole cells from the callus or in chloroplasts. The formation of putrescine by arginine decarboxylase activity gradually increased in the light until 9–12 h after subculture, whereas at the onset of the dark phase, a significant decrease was observed. Ornithine decarboxylase activity in vitro always showed slight changes, except in growing callus where putrescine synthesis increased abruptly at 8 h and decreased thereafter. Transglutaminase was immunodetected in whole cells from the callus and in isolated chloroplasts by western blot. In the entire cells, protein substrates were found which were not present in isolated chloroplast. Transglutaminase activity was light sensitive and also affected by hormone deprivation. This enzyme was more active in differentiation than in maintenance medium, in both callus and chloroplasts, in light and dark phases. These data indicate that, the parameters studied here are not only light affected but also regulated by a daily rhythm.     

Transglutaminases expression in human supraspinatus tendon ruptures and in mouse tendons

The ethiopathogenesis of rotator cuff disease remains poorly understood. Many studies advocate the importance of extra cellular matrix for the homeostasis of connective tissue. Transglutaminase enzymes family has been studied in the context of connective tissue formation and stabilisation. Here, we investigated transglutaminases expression pattern in biopsies of normal and injured supraspinatus tendons of human shoulders and in the Achilles tendons of transglutaminase 2 knock-out and wild-type mice. Our results show that different transglutaminase family members are differentially expressed in human and mouse tendons, and that transglutaminase 2 is down-regulated at mRNA and protein levels upon human supraspinatus tendon ruptures.     

Mitochondrial Aconitase is a Transglutaminase 2 Substrate: Transglutamination is a Probable Mechanism Contributing to High-Molecular-Weight Aggregates of Aconitase and Loss of Aconitase Activity in Huntington Disease Brain

Transglutaminase activity was found to be present in highly purified non-synaptosomal rat brain mitochondria. A 78-kDa protein in these organelles was shown to be a transglutaminase 2 substrate, and incubation of a non-synaptosomal mitochondrial lysate with transglutaminase 2 yielded high-M_r proteins. The 78-kDa protein was identified as mitochondrial aconitase by MALDI-TOF analysis. Aconitase activity was decreased in a dose-dependent manner when non-synaptosomal rat brain mitochondria were incubated with transglutaminase 2. Transglutaminase activity is increased about 2-fold in the mitochondrial fraction of HD caudate. Moreover, Western blotting of the mitochondrial fraction revealed that most of the mitochondrial aconitase in HD caudate is present as high-M_r aggregates. Aconitase activity was previously shown to be decreased in Huntington disease (HD) caudate (a region severely damaged by the disease). The present findings suggest that an increase of transglutaminase activity in HD caudate may contribute to mitochondrial dysfunction by incorporating aconitase into inactive polymers.     

Correlation between transglutaminase activity and polyamine levels in human neuroblastoma cells. Effect of retinoic acid and alpha-difluoromethylornithine

The human neuroblastoma cell line SK-N-BE can be induced to differentiate by retinoic acid (RA) or by alpha-difluoromethylornithine (DFMO). The former inducer produces neurite outgrowth, 60% reduction of growth rate, overexpression of neural antigens, and enhanced gamma-aminobutyric acid (GABA) and acetylcholinesterase levels. In contrast, DFMO causes cell body elongation, complete growth inhibition, and higher binding of antibodies directed against neuroectodermal antigens. Polyamine metabolism is also differently affected by the two agents. In particular a large spermine catabolism is induced by RA, while DFMO treatment leads to a small increase in the level of this compound. The neural differentiation induced by RA is accompanied by a marked increase in transglutaminase activity and its induction is paralleled by a transient increase of putrescine and spermidine. The putrescine and spermidine depletion determined by DFMO is accompanied instead by a large inhibition of transglutaminase activity. The inhibiting effect of DFMO treatment on transglutaminase is reversed by the addition of 1 mM putrescine to the culture medium. In the presence of both RA and DFMO a mixed morphological and biochemical pattern is observed. The possibility that the expression of transglutaminase associated to cellular differentiation may be modulated by the level of its substrates is also discussed.     

Transglutaminase activity in testicular homogenates and serum-free Sertoli cell cultures

Transglutaminase (EC 2.3.2.13) (TGase) activity has been localized in homogenates of rat Leydig cells and seminiferous tubules and is present in cytosol and membrane fractions. The enzyme has a requirement for Ca2+ and when the acceptor substrate casein was deleted from the assay mixture, incorporation of [14C]putrescine into cytosolic and membrane fractions occurred. Transglutaminase was also detected in Sertoli cells cultured in serum-free medium. Sertoli cells reside within the seminiferous tubule and are involved in normal spermatogenesis. Sertoli cell TGase has a strict requirement for Ca2+ and is not activated by Mg2+. Activation of the enzyme occurs with as little as 0.3 microM Ca2+; however, consistent with intracellular calcium levels, maximum stimulation occurred at 1.9 mM Ca2+. Sertoli cell TGase activity is markedly stimulated if the cells are cultured in 10% fetal bovine serum rather than in serum-free medium. Inhibition of Sertoli cell TGase by monodansylcadaverine concomitantly decreased the response of the cells to follicle-stimulating hormone (FSH)-induced secretion of cAMP but did not change basal cAMP levels. These data suggest that TGase may play a facilitative rather than an absolute role in activation of Sertoli cells by FSH and the resultant secretion of cellular products. This may occur through modulation of activities of membrane and cytosolic components by TGase.     

Epithelial distribution of neural receptors in the guinea pig small intestine

Neural and paracrine agents, such as dopamine, epinephrine, and histamine, affect intestinal epithelial function, but it is unclear if these agents act on receptors directly at the enterocyte level. The cellular localization and villus-crypt distribution of adrenergic, dopamine, and histamine receptors within the intestinal epithelium is obscure and needs to be identified. Single cell populations of villus or crypt epithelial cells were isolated from the jejunum of adult guinea pigs. Enterocytes were separated from intraepithelial lymphocytes by flow cytometry and specific binding was determined using fluorescent probes. Alpha1-adrenergic receptors were located on villus and crypt intraepithelial lymphocytes and enterocytes. Beta-adrenergic receptors were found on villus and crypt enterocytes. Dopamine receptors were found on all cell types examined, whereas histamine receptors were not detected (<10% for each cell population). These studies demonstrated that (1) receptors for epinephrine and dopamine exist on epithelial cells of the guinea pig jejunum, (2) beta-adrenergic receptors are found primarily on villus and crypt enterocytes and (3) intraepithelial lymphocytes contain alpha1-adrenergic, but have few beta-adrenergic, receptors. The presence of neural receptors suggests that these agents are acting, at least in part, at the enterocyte or intraepithelial lymphocyte levels to modulate intestinal and immune function.     

Rapid increases in the transglutaminase activity of A431 cells following treatment with epidermal growth factor

Transglutaminase activity was detected in lysates of A431 cells, a human epidermal carcinoma cell line. Enzyme activity was increased 1.5-2.5-fold in lysates prepared from cells pretreated with epidermal growth factor (EGF) relative to untreated control cells. Half-maximal activation of the transglutaminase activity occurred at 3-5 nM EGF, a concentration in good agreement with the Kd for EGF binding to its receptor in these cells. The increase in transglutaminase activity could be detected as early as 2 min after the addition of EGF, with the maximal response attained by 30 min. The activation was not blocked by pretreatment of the cells with cycloheximide, suggesting that the increased activity was not the result of an induction of transglutaminase synthesis. Fractionation of A431 cell lysates by centrifugation at 100000g for 30 min demonstrated that 90% of the transglutaminase activity was present in the soluble fraction and that this soluble transglutaminase activity was increased after treatment of the cells with EGF. The demonstration that EGF acutely increases the activity of a soluble, intracellular transglutaminase defines a novel pathway of growth factor action and provides a useful model system for identifying and comparing the mechanism(s) by which growth factors activate soluble enzymes.