Calreticulin down-regulates both GTP binding and transglutaminase activities of transglutaminase II.

Enzyme regulation is an important mechanism for controlling cell proliferation and differentiation in response to extracellular signaling molecules. We have previously reported that a approximately 50 kDa protein (termed Gbetah) consistently copurified with Galphah (transglutaminase II, TGII) and that Gbetah down-regulates the GTPase function of TGII by associating with GDP-bound TGII [Baek et al. (1996) Biochemistry 35, 2651-2657]. In this study, we examined the identity of Gbetah by partial amino acid sequencing and immunological characterizations. The results strongly suggest that Gbetah is a protein known as calreticulin (CRT). When the regulatory role of CRT in the GTPase activity of TGII was examined, CRT inhibited GTP (GTPgammaS) binding and hydrolysis in a concentration-dependent manner. Moreover, CRT interacted only with GDP-bound TGII. These results demonstrate that CRT down-regulates the GTPase activity of TGII by associating with GDP-bound TGII. Studies on the modulation of the TGase activity of TGII revealed that CRT also inhibited TGase activity. The inhibition showed the two characteristics depend on guanine nucleotides occupying the GTPase active site. The inhibition of the "empty" form of the GTPase active site increased the Ca2+ requirement without changing the Vmax. On the other hand, the inhibition of the GDP-bound form decreased Vmax, but did not alter the Ca2+ requirement. Moreover, the GTPgammaS-bound TGII was virtually resistant to Ca2+-mediated stimulation of the TGase activity, indicating that the GTP-bound TGII does not function as a TGase. We concluded that CRT is the regulatory protein of TGII that down-regulates both GTPase and TGase activities, opposing the activators of TGII function.     

GTP, an inhibitor of transglutaminases, is hydrolyzed by tissue-type transglutaminase (TGase 2) but not by epidermal-type transglutaminase (TGase 3)

Epidermal-type transglutaminase (TGase 3) is devoid of GTPase activity, but its TGase activity is inhibited by GTP as in the case of tissue-type TGase (TGase 2). In addition, the inhibition was not affected by the presence of higher concentrations of Ca ion. These results indicate that GTP interacts with TGase 3 in a manner different from its action on TGase 2.     

Structural basis for the coordinated regulation of transglutaminase 3 by guanine nucleotides and calcium/magnesium

Transglutaminase 3 (TGase 3) is a member of a family of Ca2+-dependent enzymes that catalyze covalent cross-linking reactions between proteins or peptides. TGase 3 isoform is widely expressed and is important for effective epithelial barrier formation in the assembly of the cell envelope. Among the nine TGase enzyme isoforms known in the human genome, only TGase 2 is known to bind and hydrolyze GTP to GDP; binding GTP inhibits its transamidation activity but allows it to function in signal transduction. Here we present biochemical and crystallographic evidence for the direct binding of GTP/GDP to the active TGase 3 enzyme, and we show that the TGase 3 enzyme undergoes a GTPase cycle. The crystal structures of active TGase 3 with guanosine 5'-O-(thiotriphosphate) (GTPgammaS) and GDP were determined to 2.1 and 1.9 A resolution, respectively. These studies reveal for the first time the reciprocal actions of Ca2+ and GTP with respect to TGase 3 activity. GTPgammaS binding is coordinated with the replacement of a bound Ca2+ with Mg2+ and conformational rearrangements that together close a central channel to the active site. Hydrolysis of GTP to GDP results in two stable conformations, resembling both the GTP state and the non-nucleotide bound state, the latter of which allows substrate access to the active site.     

Involucrin cross-linking by transglutaminase 1. Binding to membranes directs residue specificity

The transglutaminase 1 (TGase 1) enzyme is essential for the assembly of the cell envelope barrier in stratified squamous epithelia. It is usually bound to membranes, but to date most studies with it have involved solution assays. Here we describe an in vitro model system for characterizing the function of TGase 1 on the surface of synthetic lipid vesicles (SLV) of composition similar to eukaryote plasma membranes. Recombinant baculovirus-expressed human TGase 1 readily binds to SLV and becomes active in cross-linking above 10 microM Ca2+, in comparison to above 100 microM in solution assays, suggesting that the membrane surface is important for enzyme function. Involucrin also binds to SLV containing 12-18% phosphatidylserine and at Ca2+ concentrations above 1 microM. In reactions of involucrin with TGase 1 enzyme in solution, 80 of its 150 glutamines serve as donor residues. However, on SLV carrying both involucrin and TGase 1, only five glutamines serve as donors, of which glutamine 496 was the most favored. As controls, there was no change in specificity toward the glutamines of other substrates used by free or SLV-bound TGase 1 enzyme. We propose a model in which involucrin and TGase 1 bind to membranes shortly after expression in differentiating keratinocytes, but cross-linking begins only later as intracellular Ca2+ levels increase. Furthermore, the data suggest that the membrane surface regulates the steric interaction of TGase 1 with substrates such as involucrin to permit specific cross-linking for initiation of cell envelope barrier formation.     

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.     

Thioredoxin motif of Caenorhabditis elegans PDI-3 provides Cys and His catalytic residues for transglutaminase activity

Previous reports have suggested that protein disulfide isomerases (PDIs) have transglutaminase (TGase) activity. The structural basis of this reaction has not been revealed. We demonstrate here that Caenorhabditis elegans PDI-3 can function as a Ca(2+)-dependent TGase in assays based on modification of protein- and peptide-bound glutamine residues. By site-directed mutagenesis the second cysteine residue of the -CysGlyHisCys- motif in the thioredoxin domain of the enzyme protein was found to be the active site of the transamidation reaction and chemical modification of histidine in their motif blocked TGase activity.     

Leishmania species: evidence for transglutaminase activity and its role in parasite proliferation

Albeit transglutaminase (TGase) activity has been reported to play crucial physiological roles in several organisms including parasites; however, there was no previous report(s) whether Leishmania parasites exhibit this activity. We demonstrate herein that TGase is functionally active in Leishmania parasites by using labeled polyamine that becomes conjugated into protein substrates. The parasite enzyme was about 2- to 4-fold more abundant in Old World species than in New World ones. In L. amazonensis, comparable TGase activity was found in both promastigotes and amastigotes. TGase activity in either parasite stage was optimal at the basic pH, but the enzyme in amastigote lysates was more stable at higher temperatures (37-55 degrees C) than that in promastigote lysates. Leishmania TGase differs from mouse macrophage (M Phi) TGase in two ways: (1) the parasite enzyme is Ca(2+)-independent, whereas the mammalian TGase depends on the cation for activity, and (2) major protein substrates for L. amazonensis TGase were found within the 50-75 kDa region, while those for the M Phi TGase were located within 37-50 kDa. The potential contribution of TGase-catalyzed reactions in promastigote proliferation was supported by findings that standard inhibitors of TGase [e.g., monodansylcadaverine (MDC), cystamine (CS), and iodoacetamide (IodoA)], but not didansylcadaverine (DDC), a close analogue of MDC, had a profound dose-dependent inhibition on parasite growth. Myo-inositol-1-phosphate synthase and leishmanolysin (gp63) were identified as possible endogenous substrates for L. amazonensis TGase, implying a role for TGase in parasite growth, development, and survival.     

The multifaceted role of transglutaminase in neurodegeneration: Review article

Summary. A critical role for transglutaminase [TGase] has been hypothesized in the pathogenesis of the CAG trinucleotide repeat diseases, characterized by proteins with abnormal expansions of a polyglutamine domain. In the last few years the involvement of TGase in neurodegenerative diseases [NDS], including its role in aggregate formation, has been broadened to include Alzheimers [AD] and Parkinsons Disease [PD]. It is clear that reduction of TGase activity is beneficial for prolonged survival in mouse models of NDS. The pathological progression of these diseases might reflect in part increases of TGase induced aggregates, or changes in other pathways influenced by increases in TGase activity. Neurodegeneration may be influenced by increased TGase activity affecting apoptosis, modulation of GTPase activity and signal transduction. This review will focus on the leading hypotheses in relation to both old and new experimental results.     

Distinct pH modulation for dual function of Galphah (transglutaminase II)

Galpha(h), also known as transglutaminase II, has GTPase as well as transglutaminase activities. To better understand the factors affecting these dual enzymatic activities, we examined the optimal pH (at 25 degrees C) and thermal stability (at 37 degrees C) of the activities using membranous Galpha(h) from mouse heart. The optimum pH for the GTPase activity of Galpha(h) is approximately 7.0. As well, the GTP binding activity of Galpha(h) is more thermostable at pH 7.0 than that at pH 9.0. Consistent with these observations on the GTPase function of Galpha(h), both the phospholipase C-delta1 activity and the yield of co-immunoprecipitation of Galpha(h)-coupled phospholipase C-delta1 in alpha(1)-adrenoceptor/Galpha(h)/phospholipase C-delta1 complex preparations were enhanced by incubation with an alpha(1)-agonist, phenylephrine, at pH 7.0. On the other hand, the transglutaminase activity of Galpha(h) is higher in the basic pH range with an optimum activity at pH approximately 9.0. Also, the transglutaminase activity of Galpha(h) is more thermostable at pH 9.0 than that at pH 7.0. These results indicate not only pH as a modulator for the dual functions of Galpha(h), but also provide direct evidence for the involvement of pH in the Galpha(h)-mediated alpha(1)-adrenoceptor signaling system in vitro.     

Targeted inactivation of Gh/tissue transglutaminase II

The novel G-protein, G(h)/tissue transglutaminase (TGase II), has both guanosine triphosphatase and Ca(2+)-activated transglutaminase activity and has been implicated in a number of processes including signal transduction, apoptosis, bone ossification, wound healing, and cell adhesion and spreading. To determine the role of G(h) in vivo, the Cre/loxP site-specific recombinase system was used to develop a mouse line in which its expression was ubiquitously inactivated. Despite the absence of G(h) expression and a lack of intracellular TGase activity that was not compensated by other TGases, the Tgm2(-/-) mice were viable, phenotypically normal, and were born with the expected Mendelian frequency. Absence of G(h) coupling to alpha(1)-adrenergic receptor signaling in Tgm2(-/-) mice was demonstrated by the lack of agonist-stimulated [alpha-(32)P]GTP photolabeling of a 74-kDa protein in liver membranes. Annexin-V positivity observed with dexamethasone-induced apoptosis was not different in Tgm2(-/-) thymocytes compared with Tgm2(+/+) thymocytes. However, with this treatment there was a highly significant decrease in the viability (propidium iodide negativity) of Tgm2(-/-) thymocytes. Primary fibroblasts isolated from Tgm2(-/-) mice also showed decreased adherence with culture. These results indicate that G(h) may be importantly involved in stabilizing apoptotic cells before clearance, and in responses such as wound healing that require fibroblast adhesion mediated by extracellular matrix cross-linking.     

Distinct characteristic of Galpha(h) (transglutaminase II) by compartment: GTPase and transglutaminase activities

Galpha(h) (transglutaminase II) is a bifunctional enzyme possessing transglutaminase and GTPase activities. To better understand the factors affecting these two functions of Galpha(h), we have examined the characteristics of purified Galpha(h) from membrane and cytosol. GTP binding activity of mouse heart Galpha(h) was higher in membrane than that from cytosol. Furthermore, phospholipase C-delta1 (PLC-delta1) activity and coimmunoprecipitation of Galpha(h)-coupled PLC-delta1 in the alpha(1)-adrenoceptor-Galpha(h)-PLC-delta1 complex preparations were increased by phenylephrine in the presence of membranous Galpha(h). On the other hand, transglutaminase activity of cytosolic Galpha(h) was higher than that from membrane Galpha(h). These results demonstrate that bifunctions of Galpha(h) are regulated by its localization that can reflect the cellular functions of Galpha(h).     

Transglutaminase activity is present in highly purified nonsynaptosomal mouse brain and liver mitochondria

Several active transglutaminase (TGase) isoforms are known to be present in human and rodent tissues, at least three of which, namely, TGase 1, TGase 2 (tissue transglutaminase), and TGase 3, are present in the brain. TGase activity is known to be present in the cytosolic, nuclear, and extracellular compartments of the brain. Here, we show that highly purified mouse brain nonsynaptosomal mitochondria and mouse liver mitochondria and mitoplast fractions derived from these preparations possess TGase activity. Western blotting and experiments with TGase 2 knock-out (KO) mice ruled out the possibility that most of the mitochondrial/mitoplast TGase activity is due to TGase 2, the TGase isoform responsible for the majority of the activity ([14C]putrescine-binding assay) in whole brain and liver homogenates. The identity of the mitochondrial/mitoplast TGase(s) is not yet known. Possibly, the activity may be due to one of the other TGase isoforms or perhaps to a protein that does not belong to the classical TGase family. This activity may play a role in regulation of mitochondrial function both in normal physiology and in disease. Its nature and regulation deserve further study.     

Development of an isoenzyme-specific colorimetric assay for tissue transglutaminase 2 cross-linking activity

Tissue transglutaminase (TGase 2) belongs to the multigene transglutaminase family of Ca²⁺-dependent protein cross-linking enzymes. Based on the transamidation activity of TGase 2, a novel colorimetric assay has been developed using covalently coupled spermine to carboxy-substituted polystyrene plates and biotinylated pepT26, an excellent acyl-donor substrate, highly specific for TGase 2. The assay is based on the incorporation of the gamma-carboxamide of glutamine of pepT26 into the immobilized spermine. The amount of biotinylated pepT26 bound to the plate, as measured by the activity of streptavidin-peroxidase, is directly proportional to the TGase activity. The colorimetric procedure showed a good correlation (r = 0.995) with the commonly used radiometric filter paper method for TGase2, and provides linear dose-response curves over a wide range of hrTGase2 concentrations (2.5-40 μU/ml). In addition, the assay shows higher sensitivity when compared with our previous TG-colorimetric test (more than 50-fold increase) and other existing assays. PepT26 displays strong reactivity with TGase 2, and no reactivity with TGases 1, 3, and FXIII. The procedure constitutes a rapid, TG2-specific, sensitive, and nonisotopic method for the measurement of TGase 2 activity in as low as 4 ng of hrTGase 2 and purified guinea pig liver transglutaminase, and 1.25 μg of guinea pig liver extracts.     

Variable transglutaminase activity in human diploid fibroblasts during in vitro senescence

The specific activity of transglutaminase (TGase) was followed in human diploid fibroblasts (HDF) as a function of in vitro age. It was determined that at least 90% of the TGase activity was found in a soluble fraction at all in vitro ages; but the activity was variable with age. It was high in cells that had completed less than 50% of their lifespan (%LSC), declined to a minimum between 60 and 85% LSC, and again became elevated at more than 90% LSC. These age related variations in TGase activity could not be attributed to cellular growth characteristics, enzyme amount, or clonal selection processes. It is postulated that the variable TGase activity observed during in vitro senescence of HDF may reflect a change in affinity of the enzyme for a particular molecule; possibly fibronectin.     

Role of transglutaminase II in retinoic acid-induced activation of RhoA-associated kinase-2

Transamidation is a post-translational modification of proteins mediated by tissue transglutaminase II (TGase), a GTP-binding protein, participating in signal transduction pathways as a non-conventional G-protein. Retinoic acid (RA), which is known to have a role in cell differentiation, is a potent activator of TGASE: The activation of TGase results in increased transamidation of RhoA, which is inhibited by monodansylcadaverine (MDC; an inhibitor of transglutaminase activity) and TGaseM (a TGase mutant lacking transglutaminase activity). Transamidated RhoA functions as a constitutively active G-protein, showing increased binding to its downstream target, RhoA-associated kinase-2 (ROCK-2). Upon binding to RhoA, ROCK-2 becomes autophosphorylated and demonstrates stimulated kinase activity. The RA-stimulated interaction between RhoA and ROCK-2 is blocked by MDC and TGaseM, indicating a role for transglutaminase activity in the interaction. Biochemical effects of TGase activation, coupled with the formation of stress fibers and focal adhesion complexes, are proposed to have a significant role in cell differentiation.     

AtPng1p. The first plant transglutaminase

Studies have revealed in plant chloroplasts, mitochondria, cell walls, and cytoplasm the existence of transglutaminase (TGase) activities, similar to those known in animals and prokaryotes having mainly structural roles, but no protein has been associated to this type of activity in plants. A recent computational analysis has shown in Arabidopsis the presence of a gene, AtPng1p, which encodes a putative N-glycanase. AtPng1p contains the Cys-His-Asp triad present in the TGase catalytic domain. AtPng1p is a single gene expressed ubiquitously in the plant but at low levels in all light-assayed conditions. The recombinant AtPng1p protein could be immuno-detected using animal TGase antibodies. Furthermore, western-blot analysis using antibodies raised against the recombinant AtPng1p protein have lead to its detection in microsomal fraction. The purified protein links polyamines-spermine (Spm) > spermidine (Spd) > putrescine (Put)-and biotin-cadaverine to dimethylcasein in a calcium-dependent manner. Analyses of the gamma-glutamyl-derivatives revealed that the formation of covalent linkages between proteins and polyamines occurs via the transamidation of gamma-glutamyl residues of the substrate, confirming that the AtPng1p gene product acts as a TGase. The Ca(2+)- and GTP-dependent cross-linking activity of the AtPng1p protein can be visualized by the polymerization of bovine serum albumine, obtained, like the commercial TGase, at basic pH and in the presence of dithiotreitol. To our knowledge, this is the first reported plant protein, characterized at molecular level, showing TGase activity, as all its parameters analyzed so far agree with those typically exhibited by the animal TGases.     

Localization of cellular transglutaminase on the extracellular matrix after wounding: characteristics of the matrix bound enzyme.

Extending our previous observation that tissue transglutaminase (TGase) binds to extracellular matrix (ECM) fibronectin, we report here that endogenous tissue TGase is localized on the adjacent ECM after puncture wounding embryonic human lung fibroblasts (WI-38). The bound TGase persisted at the wound site for many hours, demonstrated by immunofluorescence and by catalytic activity using an overlay assay. The binding characteristics of TGase with ECM were studied further by the addition of exogenous TGase to cell monolayers and monitoring by immunofluorescence or overlay catalytic activity assays. Binding occurred equally well at 4 degrees C or 37 degrees C. Prior incubation of exogenous TGase with guanosine 5'-triphosphate (GTP), guanosine 5'-diphosphate (GDP), or adenosine triphosphate (ATP) had little effect on the amount bound to matrix, but prior treatment with calcium, magnesium, strontium, or manganese ions enhanced binding 2- to 3-fold. The Ca(++)-dependent change was a concentration-dependent effect on soluble exogenous TGase, rather than an effect on ECM. Immunofluorescent techniques showed that binding of exogenous TGase to ECM was prevented by prior mixing with fibronectin or collagen, but not with several other ECM components, including laminin, elastin, chondroitin sulfate, heparan sulfate, and hyaluronic acid. ECM-bound TGase was released by 2 M potassium thiocyanate (KSCN) treatment but was not released by treatment with a variety of amino acids, salts, reducing agents, glycerol, or other chaotropic agents.