Cellular transglutaminase has affinity for extracellular matrix

Summary Cellular transglutaminase (TGase) was demonstrated as an intracellular enzyme by immunofluorescence in WI-38 cells. Following cell membrane perturbation by Triton X-100 treatment, TGase was bound to the extracellular matrix and was found to coexist with fibronectin as visualized by immunofluorescence microscopy. The binding of TGase to the cell matrix was blocked by anti-fibronectin antibody. Exogenous sources of soluble TGase were transferred to the extracellular matrix of an untreated or methanol fixed cell. The experimental data indicated that “particulate bound” TGase is a consequence of soluble TGase binding to the extracellular matrix following cell rupture. Editor's statement This report suggest that “particulate bound” transglutaminase may be a consequence of affinity of soluble enzyme for specific molecules in extracellular matrix and opens up a means to characterize transglutaminase binding sites in the matrix.     

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.     

Effects of tissue transglutaminase on retinoic acid-induced cellular differentiation and protection against apoptosis.

Retinoic acid (RA) and its various synthetic analogs affect mammalian cell growth, differentiation, and apoptosis. Whereas treatment of the human leukemia cell line HL60 with RA results in cellular differentiation, addition of the synthetic retinoid, N-(4-hydroxyphenyl) retinamide (HPR), induces HL60 cells to undergo apoptosis. Moreover, pretreatment of HL60 cells as well as other cell lines (i.e. NIH3T3 cells) with RA blocks HPR-induced cell death. In attempting to discover the underlying biochemical activities that might account for these cellular effects, we found that monodansylcadaverine (MDC), which binds to the enzyme (transamidase) active site of tissue transglutaminase (TGase), eliminated RA protection against cell death and in fact caused RA to become an apoptotic factor, suggesting that the ability of RA to protect against apoptosis is linked to the expression of active TGase. Furthermore, it was determined that expression of exogenous TGase in cells exhibited enhanced GTP binding and transamidation activities and mimicked the survival advantage imparted by RA. We tested whether the ability of this dual function enzyme to limit HPR-mediated apoptosis was a result of the ability of TGase to bind GTP and/or catalyze transamidation and found that GTP binding was sufficient for the protective effect. Moreover, excessive transamidation activity did not appear to be detrimental to cell viability. These findings, taken together with observations that the TGase is frequently up-regulated by environmental stresses, suggest that TGase may function to ensure cell survival under conditions of differentiation and cell stress.     

Ca2+: a stabilizing component of the transglutaminase activity of Galphah (transglutaminase II)

Galphah (transglutaminase type II; tissue transglutaminase) is a bifunctional enzyme with transglutaminase (TGase) and guanosine triphosphatase (GTPase) activities. The GTPase function of Galphah is involved in hormonal signaling and cell growth while the TGase function plays an important role in apoptosis and in cross-linking extracellular and intracellular proteins. To analyze the regulation of these dual enzymatic activities we examined their calcium-dependence and thermal stability in enzymes from several cardiac sources (mouse heart, and normal, ischemic and dilated cardiomyopathic human hearts). The GTP binding activity of Galphah was markedly inhibited by Ca2+ whereas the TGase activity was strongly stimulated, suggesting that Ca2+ acts as a regulator, switching Galphah from a GTPase to a TGase. The TGase function of Galphah of both mouse and human hearts was more thermostable in the presence of Ca2+.     

Activation and de novo synthesis of transglutaminase in cultured glioma cells

The activity of transglutaminase (TGase) was measured in cultured C6 glioma cells after their stimulation by either isoproterenol and isobutyl-methylxanthine or by a serum-containing medium. The activity fluctuated in a biphasic manner, with the peaks at 2-3 hr and 7-8 hr poststimulation. The first peak of TGase activity was affected neither by cycloheximide nor by actinomycin D, which inhibited protein synthesis. The second peak, on the other hand, was completely eliminated by cycloheximide and was reduced by actinomycin D. Immunological procedures were employed to find out whether or not the activity of TGase corresponded with the presence of the TGase antigen in the cultured cells. Indirect immunofluorescent staining and radioimmunoblot techniques suggested that unstimulated cells contained an inactive enzyme. This inactive, or cryptic, enzyme had the same molecular weight as its active counterpart. Activation of the enzyme was mediated by cell stimulation, probably by its release from the membrane. This step did not require protein synthesis, unlike the second step, which was dependent on de novo protein synthesis.     

Transglutaminase activity and embryonal carcinoma cell differentiation

Murine embryonal carcinoma (EC) cells induced to differentiate by retinoic acid (RA) modulate transglutaminase (TGase) activity shortly after exposure to the inducer. Compounds that inhibit TGase enzyme activity in vitro can successfully block RA induced EC cell differentiation in culture. These observations suggest that TGase may play a role in mediating RA induced EC cell differentiation.     

Expression and rapid purification of highly active hexahistidine-tagged guinea pig liver transglutaminase

Tissue transglutaminase has been identified as a contributor to a wide variety of diseases, including cataract formation and Celiac disease. Guinea pig tissue transglutaminase has a very broad substrate specificity and therefore is useful for kinetic studies using substrate analogues. Here, we report the expression in Escherichia coli of a hexahistidine-tagged guinea pig liver tissue transglutaminase (His(6)-tTGase) allowing rapid purification by immobilized-metal affinity chromatography. Using this procedure we have obtained the highest reported specific activity (17 U/mg) combined with a high yield (22 mg/L of culture) for recombinant TGase using a single-step purification protocol. Using two independent spectrophotometric assays, we determined that the K(m) value of the recombinant enzyme with the substrate Cbz-Gln-Gly is in the same range as values reported in the literature for the native enzyme. We have thus developed a rapid and reproducible protocol for the preparation of high quality tissue TGase.     

转谷氨酰胺酶的分子生物学与基因工程

来源于微生物特别是轮枝链霉菌的转谷氨酰胺酶是一种重要的酶制剂,在食品工业中有着广泛的应用前景。本综述了近年来对转谷氨酰胺酶的分子生物学研究成果,以及对其进行基因工程改造的最新进展,讨论了其进一步的研究发展方向。笔认为采用基因工程生产重组转谷氨酰胺酶是解决目前酶价高昂和来源困难问题的一个大有希望的办法。     

Ordered structure acquisition by the N- and C-terminal domains of the small proline-rich 3 protein

The cell envelope (CE) is a vital structure for barrier function in terminally differentiated dead stratified squamous epithelia. It is assembled by transglutaminase (TGase) cross-linking of several proteins, including hSPR3 in certain specialized epithelia normally subjected to mechanical trauma. Biochemical studies show that hSPR3 serves as a complete substrate for TGase1, TGase2, and TGase3. Multiple adjacent glutamines and lysines of only head-and-tail domain sequences are used by each enzyme for cross-linking. Structural data suggest that the hSPR3 central repeats, as well as hSPR1 and hSPR 2, are highly flexible and mobile; thus, the TGases might not be able to recognize the residues localized on the repeats as adequate substrate. To investigate this hypothesis further and to complete the structural investigation of hSPR3, we performed circular dichroism (CD) studies on peptides corresponding to the N- and C-terminal domain. CD spectra have also been carried out in the presence of different concentrations of the structure-promoting agent cosolvent trifluoroethanol (TFE), which mimics a partial hydrophobic environment found in vivo in or next to the membrane. In fact, this agent increases the dielectric constant of water proportionally, depending on its concentration, and confers structuring properties to the solution, to peptides and proteins that have a structuring propensity. The results indicate that in both the N-terminal and C-terminal, peptides acquire a more ordered structure as a function of the TFE concentration in water. This ability of both N- and C-terminal domain to acquire a more stable ordered conformation might be relevant for SPR3 to act as substrate of TGases. Indeed, only the N- and C-terminus is cross-linked by TGase1 and 3.     

Phosphoinositide 3-kinase activity is required for retinoic acid-induced expression and activation of the tissue transglutaminase

Tissue transglutaminase (TGase) is a dual function enzyme that couples an ability to bind GTP with transamidation activity. Retinoic acid (RA) consistently induces TGase expression and activation, and it was recently shown that increased TGase expression protected cells from apoptosis. To better understand how RA regulates TGase, we considered whether RA employed pro-survival signaling pathways to mediate TGase expression and activation. It was found that RA stimulation of NIH3T3 cells activated ERK and phosphoinositide 3-kinase (PI3K); however, only PI3K activation was necessary for RA-induced TGase expression. The overexpression of a constitutively active form of PI3K did not induce TGase expression, indicating that PI3K signaling was necessary but not sufficient for TGase expression. The exposure of cells expressing exogenous TGase to the PI3K inhibitor, LY294002, reduced the ability of TGase to be photoaffinity-labeled with [alpha-(32)P]GTP, providing evidence that PI3K regulates the GTP binding activity of TGase as well as its expression. Moreover, cell viability assays showed that incubation of RA-treated cells with LY294002 together with the TGase inhibitor, monodansylcadaverine (MDC), converted RA from a differentiation factor to an apoptotic stimulus. These findings demonstrate that PI3K activity is required for the RA-stimulated expression and GTP binding activity of TGase, thereby linking the up-regulation of TGase with a well established cell survival factor.     

Cholesterol 3-sulfate interferes with cornified envelope assembly by diverting transglutaminase 1 activity from the formation of cross-links and esters to the hydrolysis of glutamine

The loss of transglutaminase 1 enzyme (TGase 1) activity causes lamellar ichthyosis. Recessive X-linked ichthyosis (XI) results from accumulation of excess cholesterol 3-sulfate (CSO(4)) in the epidermis but the pathomechanism how elevated epidermal CSO(4) causes ichthyosis is largely unknown. Here we provide evidence that XI is also a consequence of TGase 1 dysfunction. TGase 1 is a key component of barrier formation in keratinocytes: it participates in the cross-linking of cell envelope (CE) structural proteins, and also forms the lipid bound envelope by esterification of long chain omega-hydroxyceramides onto CE proteins. Using involucrin and an epidermal omega-hydroxyceramide analog as substrates, kinetic analyses revealed that at membrane concentrations above 4 mol %, CSO(4) caused a marked and dose-dependent inhibitory effect on isopeptide and ester bond formation. Sequencing of tryptic peptides from TGase 1-reacted involucrin showed a large increase in deamidation of substrate glutamines. We hypothesize that supraphysiological levels of CSO(4) in keratinocyte membranes distort the structure of TGase 1 and facilitate the access of water into its active site causing hydrolysis of substrate glutamine residues. Our findings provide further evidence for the pivotal role of the TGase 1 enzyme in CE formation.     

Activation of the Ras-ERK pathway inhibits retinoic acid-induced stimulation of tissue transglutaminase expression in NIH3T3 cells

Retinoic acid (RA) is a potent activator of tissue transglutaminase (TGase) expression, and it was recently shown that phosphoinositide 3-kinase (PI3K) activity was required for RA to increase TGase protein levels. To better understand how RA-mediated TGase expression is regulated, we considered whether co-stimulation of NIH3T3 cells with RA and epidermal growth factor (EGF), a known activator of PI3K, would facilitate the induction or increase the levels of TGase expression. Instead of enhancing these parameters, EGF inhibited RA-induced TGase expression. Activation of the Ras-ERK pathway by EGF was sufficient to elicit this effect, since continuous Ras signaling mimicked the actions of EGF and inhibited RA-induced TGase expression, whereas blocking ERK activity in these same cells restored the ability of RA to up-regulate TGase expression. However, TGase activity is not antagonistic to EGF signaling. The mitogenic and anti-apoptotic effects of EGF were not compromised by TGase overexpression, and in fact, exogenous TGase expression promoted basal cell growth and resistance to serum deprivation-induced apoptosis. Moreover, analysis of TGase expression and GTP binding activity in a number of cell lines revealed high basal TGase GTP binding activity in tumor cell lines U87 and MDAMB231, indicating that constitutively active TGase may be a characteristic of certain cancer cells. These findings demonstrate that TGase may serve as a survival factor and RA-induced TGase expression requires the activation of PI3K but is antagonized by the Ras-ERK pathway.     

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.     

Effects of Tissue Transglutaminase on β -Amyloid1-42-Induced Apoptosis

Tissue transglutaminase (TGase) has been implicated in both cell survival and apoptosis. Here we investigate the role of TGase in β-amyloid-induced neurotoxicity using retinoic acid (RA)-differentiated, neuronal SH-SY5Y cells. We show that β-amyloid-induced cell death was reduced in RA-differentiated SH-SY5Y cells treated with the TGase inhibitor monodansyl cadaverine. Expression of wild-type TGase enhanced β-amyloid_1-42-induced apoptosis, whereas transamidation-defective TGase did not. These effects were specific for β-amyloid-treated cells, as TGase reversed the neurotoxic effects caused by hydrogen peroxide treatment. Enhancement of β-amyloid_1-42-induced cell death by TGase was accompanied by marked increases in TGase activity in the membrane fractions and translocation of TGase to the cell surface. Overall, these findings suggest that the ability of TGase to exhibit pro-survival versus pro-apoptotic activity is linked to its cellular localization, with β-amyloid-induced recruitment of TGase to the cell surface accentuating neuronal toxicity and apoptosis.     

Amphotericin B inhibits the serum-induced expression of tissue transglutaminase in murine peritoneal macrophages

Culture of mouse resident peritoneal macrophages (PM) in serum-containing medium causes a rapid and marked induction of the enzyme tissue transglutaminase (tissue TGase). Coculture of PM with amphotericin B (AmpB) inhibited the serum-induced expression and accumulation of tissue TGase. The AmpB-mediated inhibition of tissue TGase was specific and was due to inhibition of enzyme synthesis. The serum-dependent induction of tissue TGase was inhibited in a dose-dependent fashion, and a complete inhibition was observed at 1.5 microgram/ml dose of AmpB. The inhibition was reversible; however, the time of recovery depended on the dose and time of exposure of the cells to AmpB. The present studies suggest that AmpB-mediated inhibition of tissue TGase is due to inhibition of the uptake of serum retinoids by PM.