"Tissue" transglutaminase in animal development

The "tissue" transglutaminase is a multifunctional enzyme that in its cross-linking configuration catalyzes Ca2+ -dependent reactions resulting in post-translational modification of proteins by establishing epsilon(gamma-glutamyl) lysine cross-links and/or covalent incorporation of biogenic amines (di- and poly-amines and histamine) into proteins. Several laboratories have shown that in Vertebrates, "tissue" transglutaminase (tTG) gene expression specifically characterizes cells undergoing apoptosis or programmed cell death (PCD). The Ca2+ -dependent activation of this enzyme leads to the formation of detergent-insoluble cross-linked protein polymers in cells undergoing PCD. This insoluble protein scaffold could stabilize the integrity of the dying cells before their clearance by phagocytosis, preventing the non-specific release of harmful intracellular components (e.g. lysosomal enzymes, nucleic acids, etc.) and consequently inflammatory responses and scar formation in bystander tissues. In this review we attempt to present an overview of the current knowledge on tTG expression and regulation in animal reproduction and development. The data available so far further strengthen the relationship existing between tTG expression and the induction of PCD.     

Tissue transglutaminase triggers oligomerization and activation of dual leucine zipper-bearing kinase in calphostin C-treated cells to facilitate apoptosis

Although tissue transglutaminase (tTG) has been recognized as a mediator of apoptosis in various experimental models, little is currently known about the molecular mechanisms by which this protein modulates cell death. Recent work from our laboratory has shown that activation of tTG in cells exposed to the apoptotic inducer calphostin C triggers the crosslinking of dual leucine zipper-bearing kinase (DLK), a proapoptotic kinase acting as an essential component of the c-Jun amino-terminal kinase (JNK) signaling pathway. As a consequence of this observation, we have undertaken experiments to investigate the functional relevance of DLK oligomerization in tTG-mediated apoptosis. Our results indicate that, in cells undergoing calphostin C-induced apoptosis, tTG-dependent DLK oligomerization occurs early in the apoptotic response. Both immunocomplex kinase assays and immunoblotting with phosphospecific antibodies revealed that oligomer formation by tTG-mediated crosslinking reactions significantly enhanced the kinase activity of DLK and its ability to activate the JNK pathway. Moreover, functional studies demonstrate that tTG-mediated oligomerization of wild-type DLK sensitizes cells to calphostin C-induced apoptosis, while crosslinking of a kinase-inactive variant of DLK does not. Collectively, these data strongly suggest that tTG facilitates apoptosis, at least partly, by oligomerization and activation of the proapoptotic kinase DLK.     

Calcium regulates S-nitrosylation, denitrosylation, and activity of tissue transglutaminase

Nitric oxide (NO) and related molecules play important roles in vascular biology. NO modifies proteins through nitrosylation of free cysteine residues, and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca(2+)-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca(2+)-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca(2+), up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca(2+), up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca(2+) to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca(2+) was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca(2+) regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca(2+) in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.     

Tissue transglutaminase is a caspase substrate during apoptosis. Cleavage causes loss of transamidating function and is a biochemical marker of caspase 3 activation

Tissue transglutaminase (tTG) is a Ca2+-dependent cross-linking enzyme that participates in the apoptotic machinery by irreversibly assembling a protein scaffold that prevents the leakage of intracellular components. In the present study a single-chain antibody fragment (scFv) detecting tTG is described. We demonstrate that TG/F8 scFv, selected from a phase display library of human V-gene segments by binding to guinea-pig liver tTG, can react with human tTG both in Western blot and in immunohistochemistry. The specific detection of tTG by TG/F8 in human thymocytes is verified by mass spectrometric analysis of the purified protein. Furthermore, we demonstrate that in lymphoid cells tTG is cleaved by caspase 3 during the late phase of apoptotic death, concomitant to DNA fragmentation, and that such cleavage causes loss of cross-linking function. We propose tTG cleavage as a valuable biochemical marker of caspase 3 activation during the late execution phase of apoptosis.     

Tissue transglutaminase cross-links beclin 1 and regulates autophagy in MPP-treated human SH-SY5Y cells

Tissue transglutaminase (tTG) is a cross-linking enzyme involved in protein aggregation during Parkinson’s disease (PD) pathogenesis. Autophagy is inhibited by tTG activation via a mechanism in which cross-linking of beclin 1, an autophagy initiator at the level of the endoplasmic reticulum (ER), has been implicated. We reported increased tTG protein levels and activity at the ER in both PD brain and in a PD-mimicking cell system. Here we characterized the interaction between tTG and beclin 1 at the ER membrane and the role of tTG in reduced autophagy in an in vitro model of PD, using differentiated SH-SY5Y neurons treated with the PD-mimic MPP⁺. We found that under PD-mimicking conditions, beclin 1 and tTG partially colocalized at the ER, beclin 1 levels increased at the ER, and tTG readily cross-linked beclin 1 which was prevented by enzymatic blockade of tTG. Under these conditions, accumulation of beclin 1 at the ER was enhanced by inhibition of tTG activity. In line with these observations and the role of beclin 1 in autophagy, levels of the autophagy marker protein LC3II in MPP⁺-treated cells, were significantly increased by inhibition of tTG activity. Our data provide first evidence for a role of tTG-mediated regulation of beclin 1 and autophagy in MPP⁺-treated human SH-SY5Y cells.     

Roles of tissue transglutaminase in ethanol-induced inhibition of hepatocyte proliferation and alpha 1-adrenergic signal transduction

The mechanisms by which ethanol inhibits hepatocyte proliferation have been a source of some considerable investigation. Our studies have suggested a possible role for tissue transglutaminase (tTG) in this process. Others have shown that tTG has two distinctly different functions: it catalyzes protein cross-linking, which can lead to apoptosis and enhancement of extracellular matrix stability, and it can function as a G protein (Galpha(h)). Under that circumstance, we speculated that the cross-linking activity would be decreased and that it would function to enhance hepatocyte proliferation in response to adrenergic stimulation. Ethanol treatment inhibited hepatocyte proliferation and led to enhanced tTG cross-linking activity, whereas treatment of hepatocytes with an alpha1 adrenergic agonist, phenylephrine, enhanced hepatocyte proliferation while decreasing tTG cross-linking. However, phenylephrine treatment of several hepatoma cell lines had no effect on cellular proliferation or tTG cross-linking activity, and of note, Northern blot analysis demonstrated that whereas primary hepatocytes had high levels of the alpha1beta adrenergic receptor (alpha1BAR) mRNA, the hepatoma cell lines did not have this mRNA. When the Hep G(2) cell line was stably transduced with an expression vector containing the alpha1BR cDNA, the cell line responded to phenylephrine treatment with enhanced proliferation and with decreased tTG cross-linking activity. Ethanol treatment of the alpha1BAR-transfected cells suppressed the phospholipase C-mediated signaling pathways, as detected in the phenylephrine-induced Ca(2+) response. These results suggest that phenylephrine stimulation of hepatocyte proliferation appears to be occurring through the alpha1BAR, which is known to be coupled with the tTG G protein moiety, Galpha(h), and that tTG appears to play a significant role in either enhancing or inhibiting hepatocyte proliferation, depending on its cellular location and on whether it functions as a cross-linking enzyme or a G protein.     

Inhibition of "tissue" transglutaminase increases cell survival by preventing apoptosis

Treatment of the human promonocytic cell line U937 with all-trans-retinoic acid (RA) commits these cells to apoptosis, which can be triggered by simply increasing intracellular calcium levels by the ionophore A23187. RA treatment of U937 cells is characterized by a decrease in Bcl-2 and marked induction of "tissue" transglutaminase (tTG) gene expression. In this study, we show that the inhibition of tTG expression in U937 cells undergoing apoptosis prevents their death. In fact, U937 cell-derived clones transfected with the human tTG gene in the antisense orientation showed a pronounced decrease in apoptosis induced by several stimuli. These findings demonstrate that the Ca(2+)-dependent irreversible cross-linking of intracellular proteins catalyzed by tTG represents an important biochemical event in the gene-regulated cell death in monoblasts. In addition, our data indicate that the apoptotic program in promonocytic cells is strictly regulated by RA and that a key role is played by the free intracellular calcium concentration.     

Developmental regulation of tissue transglutaminase in the mouse forebrain

Tissue transglutaminase (tTG) is a multifunctional enzyme that catalyzes both transamidation and GTPase reactions. In cell culture models tTG-mediated transamidation positively regulates many processes that occur in vivo during the mammalian brain growth spurt (BGS), including neuronal differentiation, neurite outgrowth, synaptogenesis and cell death mechanisms. However, little is known about the levels of tTG expression and transglutaminase (TG) activity during mammalian brain development. In this study, C57BL/6 mouse forebrains were collected at embryonic day (E) 12, E14, E17, postnatal day (P) 0, P7 and P56 and analyzed for tTG expression and TG activity. RT-PCR analysis demonstrated that tTG mRNA content increases during mouse forebrain development, whereas immunoblot analysis demonstrated that tTG protein content decreases during this time. TG activity was low in prenatal mouse forebrain but increased fivefold to peak at P0, which corresponds with the beginning of the mouse BGS. Further analysis demonstrated that the lack of temporal correlation between tTG protein content and TG activity is the result of an endogenous inhibitor of tTG that is present in prenatal but not postnatal mouse forebrain. These results demonstrate for the first time that tTG enzymatic activity in the mammalian forebrain is developmentally regulated by post-translational mechanisms.     

Tau Is Modified by Tissue Transglutaminase In Situ: Possible Functional and Metabolic Effects of Polyamination

Tissue transglutaminase (tTG) is up-regulated in Alzheimer's disease brain and localizes to neurofibrillary tangles with the tau protein. Tau is an in vitro tTG substrate, being cross-linked and/or polyaminated. Further, the Gln and Lys residues in tau that are modified by tTG in vitro are located primarily within or adjacent to the microtubule-binding domains. Considering these and other previous findings, this study was carried out to determine if tau is modified in situ by tTG in human neuroblastoma SH-SY5Y cells, and whether tTG-catalyzed tau polyamination modulates the function and/or metabolism of tau in vitro. For these studies, SH-SY5Y cells stably overexpressing tTG were used. tTG coimmunoprecipitated with tau, and elevating intracellular calcium levels with maitotoxin resulted in a 52 +/- 4% increase in the amount of tTG that coimmunoprecipitated with tau. The increase in association of tTG with tau after treatment with maitotoxin corresponded to a coimmunolocalization of tTG, tTG activity, and tau in the cells. Further, tau was modified by tTG in situ in response to maitotoxin treatment. In vitro polyaminated tau was significantly less susceptible to micro-calpain proteolysis; however, tTG-mediated polyamination of tau did not significantly alter the microtubule-binding capacity of tau. Thus, tau interacts with and is modified by tTG in situ, and modification of tau by tTG alters its metabolism. These data indicate that tau is likely to be modified physiologically and pathophysiologically by tTG, and tTG may play a role in Alzheimer's disease.     

Transglutaminase 2 activity promotes membrane resealing after mechanical damage in the lung cancer cell line A549

Transglutaminase is a Ca2+-dependent enzyme catalyzing protein cross-linking reactions. We investigated the contribution of this enzyme to the resealing of the injured plasma membrane in animal cells, using a lung cancer-derived cell line, A549. After mechanical injury by razor-scratching, the level of membrane resealing was estimated by differential incorporation of dextrans labeled with two distinct fluorescent dyes. The recovery level was decreased in the presence of excess primary amine, as a competitive inhibitor of transglutaminase. We established a cell line that stably expresses shRNA (short hairpin RNA) to specifically inhibit the expression of TGase 2 (tissue-type isozyme of transglutaminase) and confirmed the suppressed resealing level in the cell. Furthermore, additional expression of TGase 2 rescued the ability for membrane resealing. These results show that, after mechanical damage, this enzyme appeared to contribute to membrane resealing.     

Opposite effects of Ca(2+) and GTP binding on tissue transglutaminase tertiary structure

Tissue transglutaminase (tTG) belongs to a class of enzymes that catalyze a cross-linking reaction between proteins or peptides. The protein activity is known to be finely tuned by Ca(2+) and GTP binding. In this study we report the effects of these ligands on the enzyme structure, as revealed by circular dichroism, and steady-state and dynamic fluorescence measurements. We have found that calcium and GTP induced opposite conformational changes at the level of the protein tertiary structure. In particular the metal ions were responsible for a small widening of the protein molecule, as indicated by anisotropy decay measurements and by the binding of a hydrophobic probe such as 1-anilino-8-naphthalenesulfonic acid (ANS). Unlike Ca(2+), the nucleotide binding increased the protein dynamics, reducing its rotational correlation lifetime from 32 to 25 ns, preventing also the binding of ANS into the protein matrix. Unfolding of tTG by guanidinium hydrochloride yielded a three-state denaturation mechanism, involving an intermediate species with the characteristics of the so-called "molten globule" state. The effect of GTP binding (but not that of Ca(2+)) had an important consequence on the stability of tissue transglutaminase, increasing the free energy change from the native to the intermediate species by at least approximately 0.7 kcal/mol. Also a greater stability of tTG to high hydrostatic pressure was obtained in presence of GTP. These findings suggest that the molecular mechanism by which tTG activity is inhibited by GTP is essentially due to a protein conformational change which, decreasing the accessibility of the protein matrix to the solvent, renders more difficult the exposure of the active site.     

Osmotic resistance of high-density erythrocytes in transglutaminase 2-deficient mice

Transglutaminase 2 (TGase 2) is a Ca(2+)-dependent enzyme responsible for the posttransttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Elevating the intracellular concentration of Ca(2+)-ions in human erythrocytes leads to the formation of cytoskeletal and cytoplasmic protein polymers. The Ca(2+)-dependent TGase 2-dependent cross-linking activity has been proposed for its involvement in erythrocyte aging, by inducing irreversible modification of their cell shape and deformability. Accordingly, we found that high-density ("old") TGase 2(minus sign/minus sign) red blood cells (RBCs) were more resistant to osmotic stress-induced hemolysis than those from wild type mice. In addition, elevating the intracellular concentration of Ca(2+) by treatment of total RBCs with ionophore A23187 resulted in enhanced resistance of TGase 2-deficient erythrocytes compared to their normal counterpart. These findings indicate that TGase 2 may have a role in regulating structural flexibility of RBCs, possibly affecting their life span in physiopathological conditions, such as erythrocyte senescence, which are accompanied by increases in intracellular Ca(2+) concentration.     

Increase in extracellular cross-linking by tissue transglutaminase and reduction in expression of MMP-9 contribute differentially to focal segmental glomerulosclerosis in rats

Tissue transglutaminase (tTG) is a Ca²⁺-dependent enzyme which stabilizes the extracellular matrix (ECM) through post-translational modification, and may play an important role in the pathogenesis of focal and segmental glomerulosclerosis (FSGS). Here, we have investigated whether tTG contributes to the glomerular ECM expansion in the puromycin aminonucleoside (PAN)-injection-induced experimental rat model of FSGS. The localization and expression of tTG, MMP-9 gelatinase, and the ECM component fibronectin (FN) in kidneys was determined by immunohistochemistry and measured by semi-quantitative analysis. Protein levels of tTG and MMP-9 were also analyzed by Western blotting.In situtransglutaminase activity was assayed by measurement of incorporated substrate and the immunofluorescence staining for the cross-linking product, ε-(γ-glutamyl) lysine. Prominent proteinuria, a typical pathological feature of FSGS, was observed in PAN injection group rats. tTG immunoreactivity was located markedly in glomeruli and the levels of this protein in whole-kidney homogenates of PAN injection group rats were significantly increased (361± 106% control, P< 0.05). Similarly, transglutaminase activity and ε-(γ-glutamyl) lysine were also predominately located within glomeruli and were much more intense in the PAN-injected group than that in control animals. MMP-9 was also located primarily within glomeruli. In PAN-injected kidneys, protein levels of active MMP-9 were significantly reduced (59± 27% control, P< 0.01), while pro-MMP-9 levels increased (148± 42% control, P< 0.05). Remarkable expression of glomerular fibronectin (FN) was found in PAN injection group rats. Semi-quantitative analysis demonstrated this increased intensity of FN staining in the PAN-injected rats was 149± 23% of the control values (P< 0.05). Enhanced cross-linking of ECM by tissue transglutaminase and decreased degradation due to reduced active MMP-9 expression may be at least partially responsible for the deposition of FN within injured glomeruli in experimental FSGS.     

Novel protein-disulfide isomerases from the early-diverging protist Giardia lamblia.

Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum. The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions. We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia. Unlike other protein-disulfide isomerases, the giardial enzymes have only one active site. The active-site sequence motif in the giardial proteins (CGHC) is characteristic of eukaryotic protein-disulfide isomerases, and not other members of the thioredoxin superfamily that have one active site, such as thioredoxin and Dsb proteins from Gram-negative bacteria. The three giardial proteins have very different amino acid sequences and molecular masses (26, 50, and 13 kDa). All three enzymes were capable of rearranging disulfide bonds, and giardial protein-disulfide isomerase-2 also displayed oxidant and reductant activities. Surprisingly, the three giardial proteins also had Ca(2+)-dependent transglutaminase activity. This is the first report of protein-disulfide isomerases with a single active site that have diverse roles in protein cross-linking. This study may provide clues to the evolution of key functions of the endoplasmic reticulum in eukaryotic cells, protein disulfide formation, and isomerization.     

Tissue transglutaminase regulates chondrogenesis in mesenchymal stem cells on collagen type XI matrices

Tissue transglutaminase (tTG) is a multifunctional enzyme with a plethora of potential applications in regenerative medicine and tissue bioengineering. In this study, we examined the role of tTG as a regulator of chondrogenesis in human mesenchymal stem cells (MSC) using nanofibrous scaffolds coated with collagen type XI. Transient treatment of collagen type XI films and 3D scaffolds with tTG results in enhanced attachment of MSC and supports rounded cell morphology compared to the untreated matrices or those incubated in the continuous presence of tTG. Accordingly, enhanced cell aggregation and augmented chondrogenic differentiation have been observed on the collagen type XI-coated poly-(L-lactide) nanofibrous scaffolds treated with tTG prior to cell seeding. These changes implicate that MSC chondrogenesis is enhanced by the tTG-mediated modifications of the collagen matrix. For example, exogenous tTG increases resistance to collagenolysis in collagen type XI matrices by catalyzing intermolecular cross-linking, detected by a shift in the denaturation temperature. In addition, tTG auto-crosslinks to collagen type XI as detected by western blot and immunofluorescent analysis. This study identifies tTG as a novel regulator of MSC chondrogenesis further contributing to the expanding use of these cells in cartilage bioengineering.     

Tissue transglutaminase modulates alpha-synuclein oligomerization

We have studied the interaction of the enzyme tissue transglutaminase (tTG), catalyzing cross-link formation between protein-bound glutamine residues and primary amines, with Parkinson's disease-associated α-synuclein protein variants at physiologically relevant concentrations. We have, for the first time, determined binding affinities of tTG for wild-type and mutant α-synucleins using surface plasmon resonance approaches, revealing high-affinity nanomolar equilibrium dissociation constants. Nanomolar tTG concentrations were sufficient for complete inhibition of fibrillization by effective α-synuclein cross-linking, resulting predominantly in intramolecularly cross-linked monomers accompanied by an oligomeric fraction. Since oligomeric species have a pathophysiological relevance we further investigated the properties of the tTG/α-synuclein oligomers. Atomic force microscopy revealed morphologically similar structures for oligomers from all α-synuclein variants; the extent of oligomer formation was found to correlate with tTG concentration. Unlike normal α-synuclein oligomers the resultant structures were extremely stable and resistant to GdnHCl and SDS. In contrast to normal β-sheet-containing oligomers, the tTG/α-synuclein oligomers appear to be unstructured and are unable to disrupt phospholipid vesicles. These data suggest that tTG binds equally effective to wild-type and disease mutant α-synuclein variants. We propose that tTG cross-linking imposes structural constraints on α-synuclein, preventing the assembly of structured oligomers required for disruption of membranes and for progression into fibrils. In general, cross-linking of amyloid forming proteins by tTG may prevent the progression into pathogenic species.     

Does tissue transglutaminase play a role in Huntington's disease?

Tissue transglutaminase (tTG) likely plays a role in numerous processes in the nervous system. tTG posttranslationally modifies proteins by transamidation of specific polypeptide bound glutamines (Glns). This reaction results in the incorporation of polyamines into substrate proteins or the formation of protein crosslinks, modifications that likely have significant effects on neural function. Huntington's disease is a genetic disorder caused by an expansion of the polyglutamine domain in the huntingtin protein. Because a polypeptide bound Gln is the determining factor for a tTG substrate, and mutant huntingtin aggregates have been found in Huntington's disease brain, it has been hypothesized that tTG may contribute to the pathogenesis of Huntington's disease. In vitro, polyglutamine constructs and huntingtin are substrates of tTG. Further, the levels of tTG and TG activity are elevated in Huntington's disease brain and immunohistochemical studies have demonstrated that there is an increase in tTG reactivity in affected neurons in Huntington's disease. These findings suggest that tTG may play a role in Huntington's disease. However in situ, neither wild type nor mutant huntingtin is modified by tTG. Further, immunocytochemical analysis revealed that tTG is totally excluded from the huntingtin aggregates, and modulation of the expression level of tTG had no effect on the frequency of the aggregates in the cells. Therefore, tTG is not required for the formation of huntingtin aggregates, and likely does not play a role in this process in Huntington's disease brain. However, tTG interacts with truncated huntingtin, and selectively polyaminates proteins that are associated with mutant truncated huntingtin. Given the fact that the levels of polyamines in cells is in the millimolar range and the crosslinking and polyaminating reactions catalyzed by tTG are competing reactions, intracellularly polyamination is likely to be the predominant reaction. Polyamination of proteins is likely to effect their function, and therefore it can be hypothesized that tTG may play a role in the pathogenesis of Huntington's disease by modifying specific proteins and altering their function and/or localization. Further research is required to define the specific role of tTG in Huntington's disease.     

Calcium-induced intracellular cross-linking of lipocortin I by tissue transglutaminase in A431 cells. Augmentation by membrane phospholipids.

Covalently cross-linked multimers of lipocortin I are shown to be present in human epidermoid carcinoma A431 cells treated with epidermal growth factor or the calcium ionophore A23187. This intracellular cross-linking of lipocortin I is suggested to be mediated by the action of tissue transglutaminase, a Ca2(+)-dependent protein cross-linking enzyme. Cross-linking of lipocortin I competes with proteolytic digestion of the protein, and pretreatment of the cells with inhibitors for calpain (Ca2(+)-dependent intracellular protease) markedly enhanced the cross-linking of lipocortin I. Cross-linked lipocortin I is shown to be present in the soluble fraction of A431 cells as well as in the particulate fraction; a 34-kDa fragment of lipocortin I was solubilized successfully by plasmin digestion of the latter fraction. Immunofluorescence microscopy using specific antilipocortin-I antibody showed that cross-linked lipocortin I forms an envelope-like structure, which is not extracted with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) or Triton X-100. In vitro incubation of purified lipocortin I with tissue transglutaminase resulted in the formation of covalently cross-linked lipocortin I dimer, tetramer, and so on. Amine incorporation and cross-linking studies using lipocortin I and its N-terminal truncated derivatives indicated that the cross-linking site is localized within the plasmin-susceptible N-terminal 29 amino acids of lipocortin I. The cross-linking of lipocortin I is shown to be accelerated more than 10 times by the addition of phosphatidylserine vesicles, on which lipocortin I molecules are most likely aligned in a conformation suitable for cross-linking. Collectively, these findings suggest that an increase of intracellular calcium concentration results in the attachment of lipocortin I onto the plasma membrane phospholipids through the C-terminal domain of the molecule where the membrane-bound lipocortin I is cross-linked by the action of tissue transglutaminase through the N-terminal domain.     

Tissue transglutaminase expression in quail epiphyseal chondrocytes.

Tissue transglutaminase (tTGase) is a GTP-binding Ca(2+)-dependent enzyme which catalyses the post-translational modification via epsilon(gamma-glutamyl)lysine bridges. The physiological role of tTGase is not fully understood. It has been shown that in cartilage the expression of tTGase correlates with terminal differentiation of chondrocytes. Recent evidence suggests that the GTP-binding activity of tTGase may play a role in the control of cell cycle progression thus explaining some of the suggested roles for the enzyme.tTGase activity is present in primary cultures of epiphyseal chondrocytes and increases transiently upon retinoic acid (RA) treatment. Increase in enzyme activity occurs upon RA addition and is accompanied by a parallel increase in protein and mRNA levels. Stimulation of tTGase expression by RA correlates with suppression of cell growth and occurs independently of cell adhesion and cell differentiation.tTGase expression is not observed in MC2, a permanent chondrocyte cell line derived from retrovirus infected chondrocytes. RA treatment fails to activate tTGase expression in MC2 cells and to completely suppress cell proliferation.Our findings lend support to the idea that tTGase might play a role in non-dividing cultured chondrocytes.