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Increased transglutaminase activity during skin wound healing in rats
Abstract
Outer, middle and inner layers from wounded or unwounded rat dorsal skin were separated and extracted first with buffer and then with Triton X-100 and dithiothreitol. The extracts and residues were assayed for transglutaminase activity and tissue transglutaminase antigen. Transglutaminase activities in all skin layers are increased in the period 1-5 days after wounding. Most of the increased activity is in the buffer-soluble fraction in the inner skin layer though there is no corresponding increase in antigen in this fraction. This suggests that there is production of activated soluble tissue transglutaminase in the wounded inner layer. In the 3-5 day wounded outer layer the largest fraction of both activity and antigen is associated with the insoluble residue remaining after extraction with Triton X-100. On DEAE-cellulose chromatography Triton X-100 extracts of the inner layer of wounded skin showed a single major peak of activity, corresponding approximately with rabbit liver transglutaminase; the outer layer showed the same peak plus a different one, eluting at lower salt concentration, which is thought to be epidermal transglutaminase.
... The ability of tTGase to create covalent protein cross-links suggests its involvement in maintaining tissue integrity; and as a consequence, the enzyme is thought to play an important role in various physiological as well as pathological situations such as wound healing, fibrosis, inflammation, and tumor metastasis (7)(8)(9)(10)(11). Although tTGase was originally thought to be an intracellular enzyme, accumulating evidence indicates that the enzyme is externalized and capable of cross-linking a wide range of extracellular matrix (ECM) proteins, which is thought to be important in ECM deposition/stabilization and the cell attachment and spreading of a number of different cell types (12)(13)(14)(15). ...
... Because tTGase is involved in both cell attachment and spreading and, through its cross-linking activity, in wound healing and tissue fibrosis (7)(8)(9)(10)(11)28), it is not unreasonable to assume that it might also be involved in cell migration, a process that is important to a number of cellular events, including embryogenesis, tissue repair, and tumor invasion. To explore this, we have used 3T3 fibroblasts transfected with a number of different tTGase constructs expressing the catalytically active or inactive forms of tTGase. ...
Increasing evidence suggests that tissue transglutaminase (tTGase; type II) is externalized from cells, where it may play a key role in cell attachment and spreading and in the stabilization of the extracellular matrix (ECM) through protein cross-linking. However, the relationship between these different functions and the enzyme's mechanism of secretion is not fully understood. We have investigated the role of tTGase in cell migration using two stably transfected fibroblast cell lines in which expression of tTGase in its active and inactive (C277S mutant) states is inducible through the tetracycline-regulated system. Cells overexpressing both forms of tTGase showed increased cell attachment and decreased cell migration on fibronectin. Both forms of the enzyme could be detected on the cell surface, but only the clone overexpressing catalytically active tTGase deposited the enzyme into the ECM and cell growth medium. Cells overexpressing the inactive form of tTGase did not deposit the enzyme into the ECM or secrete it into the cell culture medium. Similar results were obtained when cells were transfected with tTGase mutated at Tyr(274) (Y274A), the proposed site for the cis,trans peptide bond, suggesting that tTGase activity and/or its tertiary conformation dependent on this bond may be essential for its externalization mechanism. These results indicate that tTGase regulates cell motility as a novel cell-surface adhesion protein rather than as a matrix-cross-linking enzyme. They also provide further important insights into the mechanism of externalization of the enzyme into the extracellular matrix.
... These reports demonstrated that tTgase is important for controlling matrix storage of LTBP-1, therefore regulating TGF-b1 availability and activation that has important consequences for tissue repair and wound healing (Border and Ruoslahti, 1992;Nurminskaya et al, 1998). In this respect, increasing numbers of reports support a role for tTgase as an important factor in wound repair and ¢brosis (Bowness et al, 1988;Upchurch et al, 1991;Dolynchuk et al, 1996;Raghunath et al, 1996;Johnson et al, 1997Johnson et al, , 1999Haroon et al, 1999). Another important function for tTgase relates to its role as an e¡ector protein during cell death. ...
... From a more physiologic context we have also observed this increase in matrix associated tTgase activity without increase in enzyme expression in the tubulo-interstitium of rat kidney during renal scarring for hyperglycemia (Skill et al, 2001). A comparable observation was also demonstrated by Bowness et al (1988) following the mechanical wounding of rat skin. ...
Investigations were undertaken to study the role of the protein cross-linking enzyme tissue transglutaminase in changes associated with the extracellular matrix and in the cell death of human dermal fibroblasts following exposure to a solarium ultraviolet A source consisting of 98.8% ultraviolet A and 1.2% ultraviolet B. Exposure to nonlethal ultraviolet doses of 60 to 120 kJ per m2 resulted in increased tissue transglutaminase activity when measured either in cell homogenates, "in situ" by incorporation of fluorescein-cadaverine into the extracellular matrix or by changes in the epsilon(gamma-glutamyl) lysine cross-link. This increase in enzyme activity did not require de novo protein synthesis. Incorporation of fluorescein-cadaverine into matrix proteins was accompanied by the cross-linking of fibronectin and tissue transglutaminase into nonreducible high molecular weight polymers. Addition of exogenous tissue transglutaminase to cultured cells mimicking extensive cell leakage of the enzyme resulted in increased extracellular matrix deposition and a decreased rate of matrix turnover. Exposure of cells to 180 kJ per m2 resulted in 40% to 50% cell death with dying cells showing extensive tissue transglutaminase cross-linking of intracellular proteins and increased cross-linking of the surrounding extracellular matrix, the latter probably occurring as a result of cell leakage of tissue transglutaminase. These cells demonstrated negligible caspase activation and DNA fragmentation but maintained their cell morphology. In contrast, exposure of cells to 240 kJ per m2 resulted in increased cell death with caspase activation and some DNA fragmentation. These cells could be partially rescued from death by addition of caspase inhibitors. These data suggest that changes in cross-linking both in the intracellular and extracellular compartments elicited by tissue transglutaminase following exposure to ultraviolet provides a rapid tissue stabilization process following damage, but as such may be a contributory factor to the scarring process that results.
... The role of TG2 in cellular processes that are relevant to wound healing, such as cell adhesion, spreading and migration, has been widely studied and well-reviewed [24,25]. TG2 expression and activity are increased at sites of neovascularization and invasion of the fibrin matrix and, later, in the granulation tissue matrix where it may crosslink ECM substrates [26][27][28]. Studies of cultured cells have provided evidence for a role of TG2 in adhesion, spreading and motility [18,23,[29][30][31][32]. ...
Transglutaminase 2 (TG2) plays a role in cellular processes that are relevant to wound healing, but to date no studies of wound healing in TG2 knockout mice have been reported. Here, using 129T2/SvEmsJ (129)- or C57BL/6 (B6)-backcrossed TG2 knockout mice, we show that TG2 facilitates murine wound healing in a strain-dependent manner. Early healing of in vivo cutaneous wounds and closure of in vitro scratch wounds in murine embryonic fibroblast (MEF) monolayers were delayed in 129, but not B6, TG2 knockouts, relative to their wild-type counterparts, with wound closure in 129 being faster than in B6 wild-types. A single dose of exogenous recombinant wild-type TG2 to 129 TG2-/- mice or MEFs immediately post-wounding accelerated wound closure. Neutrophil and monocyte recruitment to 129 cutaneous wounds was not affected by Tgm2 deletion up to 5 days post-wounding. Tgm2 mRNA and TG2 protein abundance were higher in 129 than in B6 wild-types and increased in abundance following cutaneous and scratch wounding. Tgm1 and factor XIIA (F13A) mRNA abundance increased post-wounding, but there was no compensation by TG family members in TG2-/- relative to TG2+/+ mice in either strain before or after wounding. 129 TG2+/+ MEF adhesion was greater and spreading was faster than that of B6 TG2+/+ MEFs, and was dependent on syndecan binding in the presence, but not absence, of RGD inhibition of integrin binding. Adhesion and spreading of 129, but not B6, TG2-/- MEFs was impaired relative to their wild-type counterparts and was accelerated by exogenous addition or transfection of TG2 protein or cDNA, respectively, and was independent of the transamidase or GTP-binding activity of TG2. Rho-family GTPase activation, central to cytoskeletal organization, was altered in 129 TG2-/- MEFs, with delayed RhoA and earlier Rac1 activation than in TG2+/+ MEFs. These findings indicate that the rate of wound healing is different between 129 and B6 mouse strains, correlating with TG2 abundance, and although not essential for wound healing, TG2 facilitates integrin- and syndecan-mediated RhoA- and Rac1-activation in fibroblasts to promote efficient wound contraction.
... Bowness et al. (16,17) reported TG activity was present at sites of wound healing and that inhibition of TG by putrescine caused decreased breaking strength and increased solubility of the repairing wound tissue (18). However, the cellular expression, distribution, and metabolic fate of the TG at sites of wound healing in relationship to the expression of cytokines and migration of inflammatory cells were not investigated. ...
... 76 Such injury also results in a local release of the intracellular enzyme, tissue transglutaminase (tTG). This enzyme comes mainly from fibroblasts and endothelial cells 77 and has a key role in the maintenance of tissue stability (cross-links several matrix proteins and thus stabilizes the scaffold of connective tissues). 78 Transglutaminase crosslinks and deamidates gluten and thus enhances the binding of gliadin peptides to the HLA DQ2 and 8 molecules on leucocytes. ...
... Transglutaminases (TGs) comprise a family of Ca 2?dependent enzymes that catalyze protein cross-linking between glutamine and lysine residues (Jeitner et al. 2009;Kornguth and Waelsch 1963), which are involved in various physiological processes in mammals, including blood coagulation, terminal differentiation of epidermal keratinocytes, wound healing, cell signaling, survival, and apoptosis (Lorand et al. 1966;Rice and Green 1978;Bowness et al. 1988;Boroughs et al. 2014;Fesus et al. 1987). There are nine TG family members in humans, i.e., TG1, TG2, TG3, TG4, TG5, TG6, TG7, Factor XIII, and EPB42, whereas Drosophila contains only a single homolog, Tg (CG7356) (Esposito and Caputo 2005;Bakker et al. 2008). ...
Transglutaminases (TGs) comprise a family of Ca(2+)-dependent enzymes that catalyze protein cross-linking, which include nine family members in humans but only a single homolog in Drosophila with three conserved domains. Drosophila Tg plays important roles in cuticle morphogenesis, hemolymph clotting, and innate immunity. Mammalian tissue TG (TG2) is involved in polyglutamine diseases (polyQ diseases), and TG6 has been identified as a causative gene of a novel spinocerebellar ataxia, SCA35. Using a well-established SCA3 fly model, we found that RNA interference-mediated suppression of Tg aggravated polyQ-induced neurodegenerative phenotypes. The administration of cystamine, a known effective Tg inhibitor, enhanced ommatidial degeneration in SCA3 flies. We also demonstrated that the aggregates of pathogenic ataxin-3 increased greatly, when the Tg activity was repressed. These findings indicate that Tg is crucial for polyQ-induced neurotoxicity because Tg ablation resulted in more severe neurodegeneration due to the elevated accumulation of insoluble ataxin-3 complexes in the SCA3 Drosophila model.
... The profiles of neutrophil and monocyte infiltration into wounds of TG2 -/mice were the same as their wildtype counterparts and no significant differences were observed between TG2 +/+ and TG2 -/mice at any time point for either population (Figure 11c-g). These findings differ from the observations that macrophage infiltration was positively correlated with TG2 protein expression in a skin injury study [238] or decreased neutrophil recruitment to kidneys with endotoxic shock in TG2-deficient mice [239]. ...
... TG2's transglutaminase activity has an important role in would healing, and the enzyme is elevated in healing wounds in cell culture 46 and in vivo. 92,93 Initially, TG2 activity is a result of activation of previously inactive enzyme, not synthesis of new protein, but smooth muscle cells, fibroblasts, and myofibroblasts all synthesize new TG2 in the ECM of the healing wound. 94,95 TG2 stabilizes the matrix forming in the wound as cells move in and replace the original clot matrix (itself stabilized by Factor XIIIa crosslinking). ...
Ageing of the extracellular matrix (ECM), the protein matrix that surrounds and penetrates the tissues and binds the body together, contributes significantly to functional aging of tissues. ECM proteins become increasingly cross-linked with age, and this cross-linking is probably important in the decline of the ECM's function. In this paper I review the role of EGGL, a cross-link formed by transglutaminase enzymes, and particularly the widely expressed isozyme TG2, in the aging ECM. There is little direct data on EGGL accumulation with age, and no direct evidence of a role of EGGL in the aging of the ECM outwith pathology. However, several lines of circumstantial evidence suggest that EGGL accumulates with age, and its association with pathology suggests that this might reflect degradation of ECM function. TG activity increases with age in many circumstances, ECM protein turnover is such that some EGGL made by TG is likely to remain in place for years if not decades in healthy tissue, and both EGGL and TG levels are enhanced by age-related diseases. If further research shows EGGL does accumulate with age, removing it could be of therapeutic benefit. I review blockade of TG and active removal of EGGL as therapeutic strategies, and conclude that both have promise. EGGL removal may have benefit for acute fibrotic diseases such as tendinopathy, and for treating generalized decline in ECM function with old age. Extracellular TG2 and EGGL are therefore therapeutic targets both for specific and more generalized diseases of aging.
... The importance of tTG in wound healing was initially based on the presence of defective wound healing in factor XIII deficiency. Bowness et al described the presence of tTG in wound healing in rats and made the important observation that although antigen and activity both increased, there was more antigen than activity indicating degradation or inactivation of the protein [1]. Application of putrescine, a transglutaminase inhibitor, produced a significant decrease in wound strength between day 5 and 10 [2]. ...
Angiogenesis is vital to tumor growth and metastasis. The scope of this grant was to study in detail the role of Tissue transgiutaminase (tTG) during wound healing and tumorigenesis. In the first year of the grant proposal, we have described the expression, localization, molecular form and tTG's association with other major determinants of wound healing and tumorigenesis. Our findings clearly show that tTG is readily unregulated in wound healing and rat mammary adenocarcinoma and is associated with endothelial and inflammatory cells. Hypoxia, Vascular endothelial growth factor, Transforming growth factor heta and Tumor necrosisi factor alpha are also unregulated alongside tTG in those cells. tTG is quickly proteolysed in the tissues and that may have important consequences as tTG can hydrolyze ATP/GTP in its fragmented form. We are submitting these findings to Journal of Clinical investigation Proceedings of National Academy of Sciences and American Journal of Pathology where we have detailed the potential significance of these observations. We have also addressed most of the tasks detailed in the statement of work and are now set to proceed with the rest of the project.
... This suggests the “activated” keratinocytes seen in wound healing changed in the absence of TGase 3. These cells produce increased levels of transglutaminases and growth factors [51]. TGase 3, like factor XIII and TGase 2, is released from cells [16], [39] and may play a role in TGFβ activation [52], fibronectin signaling [53], and cross-linking of the extracellular matrix [29], [54]. ...
Transglutaminases (TGase), a family of cross-linking enzymes present in most cell types, are important in events as diverse as cell-signaling and matrix stabilization. Transglutaminase 1 is crucial in developing the epidermal barrier, however the skin also contains other family members, in particular TGase 3. This isoform is highly expressed in the cornified layer, where it is believed to stabilize the epidermis and its reduction is implicated in psoriasis. To understand the importance of TGase 3 in vivo we have generated and analyzed mice lacking this protein. Surprisingly, these animals display no obvious defect in skin development, no overt changes in barrier function or ability to heal wounds. In contrast, hair lacking TGase 3 is thinner, has major alterations in the cuticle cells and hair protein cross-linking is markedly decreased. Apparently, while TGase 3 is of unique functional importance in hair, in the epidermis loss of TGase 3 can be compensated for by other family members.
... Mallory body inclusions consisting primarily of tranglutaminase cross-linked keratins 8 and 18 are characteristic of several liver disorders, although it is not yet clear whether these bodies protect from, or promote, injury [109]. In response to cutaneous injury, TG2 expression and activity are increased at sites of neovascularization in the provisional fibrin matrix, endothelial cells, skeletal muscle cells, and macrophages infiltrating wounds in the border zone between normal and injured tissue and, later, in the granulation tissue matrix where it is thought to cross-link ECM substrates [110,111]. Recent studies using lipopolysaccaride to induce murine endotoxic shock demonstrated increased survival of C57BL/6 TG2-deficent mice relative to their wild-type counterparts [112]. This was associated with decreased NFkB activation, decreased neutrophil recruitment into kidneys and peritoneum, and reduced renal and myocardial damage. ...
Introduction Biochemistry of TG2 Molecular Biology of TG2 Physiopathology of TG2 Medical Perspectives and Future Directions Acknowledgments References
... Transglutaminase enzymes are expressed in a variety of cell types and have the natural function to crosslink several ECM proteins [10,51]. Over traditional chemical crosslinking or gelation agents, transglutaminase enzymes have the advantages to be employed under mild conditions (such as temperature and pH) and not requiring chemical initiators. ...
Living organisms synthesize functional materials, based on proteins and polysaccharides, using enzyme-catalyzed reactions. According to the biomimetic approach, biomaterial matrices for tissue engineering are designed to be able to mimic the properties and the functions of the extracellular matrix (ECM). In this chapter, the most significant research efforts dedicated to the study and the preparation of biomimetic materials through enzymatic modifications were reviewed. The functionalizations of different polymeric matrices obtained through the catalytic activity of two enzymes (Transglutaminase, TGase and Tyrosinase, TYRase) were discussed. Specifically, the biomimetic applications of TGase and TYRase to confer appropriate biomimetic properties to the biomaterials, such as the possibility to obtain in situ gelling hydrogels and the incorporation of bioactive molecules (growth factors) and cell-binding peptides into the scaffolds, were reviewed.
Graphical Abstract
... Mallory body inclusions consisting primarily of transglutaminase cross-linked keratins 8 and 18 are characteristic of several liver disorders, although it is not yet clear whether these bodies protect from, or promote, injury (310). In response to cutaneous injury, TG2 expression and activity is increased at sites of neovascularization in the provisional fibrin matrix, endothelial cells, skeletal muscle cells, and macrophages infiltrating wounds in the border zone between normal and injured tissue, and, later, in the granulation tissue matrix where it is thought to cross-link ECM substrates (49,109). ...
The human transglutaminase (TG) family consists of a structural protein, protein 4.2, that lacks catalytic activity, and eight zymogens/enzymes, designated factor XIII-A (FXIII-A) and TG1-7, that catalyze three types of posttranslational modification reactions: transamidation, esterification, and hydrolysis. These reactions are essential for biological processes such as blood coagulation, skin barrier formation, and extracellular matrix assembly but can also contribute to the pathophysiology of various inflammatory, autoimmune, and degenerative conditions. Some members of the TG family, for example, TG2, can participate in biological processes through actions unrelated to transamidase catalytic activity. We present here a comprehensive review of recent insights into the physiology and pathophysiology of TG family members that have come from studies of genetically engineered mouse models and/or inherited disorders. The review focuses on FXIII-A, TG1, TG2, TG5, and protein 4.2, as mice deficient in TG3, TG4, TG6, or TG7 have not yet been reported, nor have mutations in these proteins been linked to human disease.
... TG is suggested to be involved in matrix maturation and stabilize the tissue with cross-links that are resistant to normal proteolysis (1,2). TG is closely related to wound healing which suggests a role for it in tissue remodeling and repair (3,4). Immunohistochemical data have also demonstrated the presence of TG in mineralizing cartilage and bone (5,6) and the enzyme is thought to participate in matrix cross-linking before the tissue undergoes calcification (5,6). ...
Osteopontin, a major noncollagenous bone protein, is an in vitro and in vivo substrate of tissue transglutaminase, which catalyzes formation of cross-linked protein aggregates. The roles of the enzyme and the polymeric osteopontin are presently not fully understood. In this study we provide evidence that transglutaminase treatment significantly increases the binding of osteopontin to collagen. This was tested with an enzyme-linked immunosorbent assay. The results also show that this increased interaction is clearly calcium-dependent and specific to osteopontin. In dot blot overlay assay 1 microgram of collagen type I was able to bind 420 ng of in vitro prepared and purified polymeric osteopontin and only 83 ng of monomeric osteopontin, indicating that the transglutaminase treatment introduces a 5-fold amount of osteopontin onto collagen. Assays using a reversed situation showed that the collagen binding of the polymeric form of osteopontin appears to be dependent on its conformation in solution. Circular dichroism analysis of monomeric and polymeric osteopontin indicated that transglutaminase treatment induces a conformational change in osteopontin, probably exposing motives relevant to its interactions with other extracellular molecules. This altered collagen binding property of osteopontin may have relevance to its biological functions in tissue repair, bone remodeling, and collagen fibrillogenesis.
... In addition to its ability to stabilize the plaque by cross-linking important ECM substrates, TG has other roles that could affect the stability of the atheromatous plaque and its response to injury. These include a direct cell adhesive action (Gentile et al, 1992), roles in apoptosis (Fesus et al, 1989(Fesus et al, , 1991, the regulation of cell growth (Birckbichler and Patterson, 1978), and wound healing (Bowness et al, 1988). TG in the intracellular compartment also has a recently recognized role as a G protein in signal transduction (Nakaoka et al, 1994). ...
Although atherosclerosis progresses in an indolent state for decades, the rupture of plaques creates acute ischemic syndromes that may culminate in myocardial infarction and stroke. Mechanical forces and matrix metalloproteinase activity initiate plaque rupture, whereas tissue inhibitors of metalloproteinases have an important (albeit indirect) role in plaque stabilization. In this paper, an enzyme that could directly stabilize the plaque is described. Tissue transglutaminase (TG) catalyzes the formation of epsilon(gamma-glutamyl)lysine isopeptide bonds that are resistant to enzymatic, mechanical, and chemical degradation. We performed immunohistochemistry for TG in atherosclerotic human coronary and carotid arteries. TG was most prominent along the luminal endothelium and in the medium of the vessels with a distribution mirroring that of smooth muscle cells. Variable, often prominent, immunoreactivity for TG was also seen in the intima, especially in regions with significant neovascularization. Additionally, TG was detected in fibrous caps and near the "shoulder regions" of some plaques. A monoclonal antibody to the transglutaminase product epsilon(gamma-glutamyl)lysine isopeptide demonstrated co-localization with TG antigen. Transglutaminase activity was found in 6 of 14 coronary artery atherectomy samples. Cross-linking of TG substrates such as fibrinogen, fibronectin, vitronectin, collagen type I, and protease inhibitors stabilized the plaque. Furthermore, the activation of transforming growth factor-beta-1 by TG might be an additional mechanism for the promotion of plaque stabilization and progression by increasing the synthesis of extracellular matrix components.
... 8 Further, transglutaminase (t-TGase), a kind of cell adhesion molecules, is a calciumdependent enzyme that cross-links the glutamate and lysine residue of two polypeptides. 9 The t-TGase has been suggested to be associated with the regulation of cell proliferation, differentiation, and wound healing. 10 It is also implicated in the modulation of cell apoptosis. ...
SACCHACHITIN membrane, a weavable skin substitute made from the residual fruiting body of Ganoderma tsugae, has been demonstrated to promote skin wound healing. Prior to its clinical application, it is critical to learn more about any possible cytotoxicity, immunogenicity, or allergy response, and at least some of its mechanism(s) of action(s). In the present studies, it has been found that SACCHACHITIN suspension at less than 0.05% shows no cytotoxicity to the primary culture of rat fibroblasts. However, at higher concentrations (> or = 0.1%), it does reduce the growth of fibroblasts, based on MTT assays. This might be caused by positive charges on chitin molecules that are too strong, and may be harmful to the cell membrane. SACCHACHITIN showed no immunogenicity after it was inoculated into rats three times; however, the unmodified, purified rabbit type I and type II collagens did. Subcutaneous injection of SACCHACHITIN suspension into rats showed no gross allergic responses on skin. Nevertheless, it did cause local acute inflammation, as observed by histological investigation. This is similar to what occurred in the wound site covered with SACCHACHITIN membrane. The chemotactic effect of SACCHACHITIN was exhibited in both intact and wounded skin tissues. This may be one of the initial beneficial effects of SACCHACHITIN membrane to wound healing. The rapid acute inflammatory process was followed by the appearance of angiogenesis and granulation tissue formation, which occurred earlier than it normally would. Coverage of the wound area with SACCHACHITIN membrane also induced an earlier formation of scar tissue to replace the granulation tissue. A 1.5 x 1.5 cm(2) wound area covered by SACCHACHITIN completely healed by 21 days, while that covered with cotton gauze did not. Therefore, SACCHACHITIN is a safe biomaterial for use as a wound dressing for skin healing. Its promoting action for wound healing might be due to its chemotactic effect for inflammatory cells. This, in turn, may facilitate subsequent angiogenesis, granulation tissue formation, and faster new tissue formation, leading to faster wound healing.
... However, there is now increasing evidence to suggest that tTG can act at the cell surface facilitating cell adhesion, (5,6) cell spreading, (7,8) and the modification of the ECM. (9 -12) tTG also has been implicated in programmed cell death, (13) wound healing, (11) tumor growth, (14) and in the bone mineralization process. (15) It is not yet known which TG is expressed by osteoblasts in vitro. ...
Tissue transglutaminase (tTG) is a calcium-dependent and guanosine 5'-triphosphate (GTP) binding enzyme, which catalyzes the post-translational modification of proteins by forming intermolecular epsilon(gamma-glutamyl)lysine cross-links. In this study, human osteoblasts (HOBs) isolated from femoral head trabecular bone and two osteosarcoma cell lines (HOS and MG-63) were studied for their expression and localization of tTG. Quantitative evaluation of transglutaminase (TG) activity determined using the [1,4 14C]-putrescine incorporation assay showed that the enzyme was active in all cell types. However, there was a significantly higher activity in the cell homogenates of MG-63 cells as compared with HOB and HOS cells (p < 0.001). There was no significant difference between the activity of the enzyme in HOB and HOS cells. All three cell types also have a small amount of active TG on their surface as determined by the incorporation of biotinylated cadaverine into fibronectin. Cell surface-related tTG was further shown by preincubation of cells with tTG antibody, which led to inhibition of cell attachment. Western blot analysis clearly indicated that the active TG was tTG and immunocytochemistry showed it be situated in the cytosol of the cells. In situ extracellular enzyme activity also was shown by the cell-mediated incorporation of fluorescein cadaverine into extracellular matrix (ECM) proteins. These results clearly showed that MG-63 cells have high extracellular activity, which colocalized with the ECM protein fibronectin and could be inhibited by the competitive primary amine substrate putrescine. The contribution of tTG to cell surface/matrix interactions and to the stabilization of the ECM of osteoblast cells therefore could by an important factor in the cascade of events leading to bone differentiation and mineralization.
... 1,2 tTG activity has been demonstrated to play a role in stabilization of the basement membrane and adhesion of cells, 3,4 which are important processes in wound healing, angiogenesis, and bone remodeling. [5][6][7] In particular, tTG has been shown to cross-link components of the extracellular matrix (ECM), including fibronectin, vitronectin, laminin, and collagen. 8 -12 tTG is expressed by a variety of cell types, 1,2 including endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages, which (along with fibrin) are major components of atherosclerotic lesions. ...
Atherosclerosis is characterized by thickening of the vessel wall, smooth muscle cell proliferation, macrophage infiltration, and deposition of a fibrin network. Transglutaminases are a family of enzymes catalyzing the formation of stable covalent cross-links between proteins. Here, we show that tissue transglutaminase (tTG) synthesis by human umbilical vein endothelial cells is upregulated by thrombin, the serine protease that causes fibrin formation and many cellular inflammatory effects. Thrombin upregulated tTG 2-fold at the mRNA and protein level. Cellular cross-linking activity was increased to an even greater extent; antibody to tTG neutralized the increased activity. The effect on tTG expression required active thrombin and was mediated mainly through protease-activated receptor-1, a thrombin receptor. Increased tTG antigen and activity were evident in human umbilical vein endothelial cells and extracellular matrix in situ. Thrombin treatment also led to a cellular redistribution of tTG. Normal vessel wall stained positively for tTG in the smooth muscle cells and in the subendothelium. The intensity of staining increased in vessel walls with plaque, where there was a striking increase in tTG in the smooth muscle cells immediately below the plaque. These studies indicate a role for tTG in the stabilization of atherosclerotic plaques and suggest that its local expression can be controlled by thrombin.
... Tissue transglutaminase also appears to be released from connective tissue cells and to become tightly associated with the extracellular matrix (Aeschlimann and Paulsson, 1991;Aeschlimann et al, 1993). The transglutaminase-mediated cross-linking of matrix proteins contributes to the mechanical properties of the specialized matrices involved in osteogenesis Paulsson, 1991, 1994), angiogenesis (Barsigian et al, 1991) and wound healing (Bowness et al, 1988). ...
Tissue transglutaminase is a multifunctional enzyme that accumulates to high levels in cells undergoing apoptosis. Retinoids act as an acute and direct regulator of tissue transglutaminase gene transcription. The studies reported here were carried out to elucidate the molecular mechanisms involved in the regulation of tissue transglutaminase expression. We have isolated and characterized the mouse tissue transglutaminase gene promoter and 3.8 kb of 5'-flanking DNA. A large fragment of the promoter that includes both the core promoter and 3.8 kb of 5'-flanking DNA shows retinoid-dependent transcriptional activity when stably transfected into HeLa cells. In these stably transfected HeLa cells both the endogenous tissue transglutaminase gene and transfected mouse tissue transglutaminase promoter are activated by all-trans retinoic acid and by retinoic acid receptor (RAR)-specific and retinoid X receptor (RXR)-specific retinoids. In embryos made transgenic with a transglutaminase promoter-beta-galactosidase reporter gene, the transgene shows specific patterns of expression during limb development. The transglutaminase transgene is expressed in cartilage, the cells of the apical ectodermal ridge, and in regions of apoptotic cell death of the interdigital mesenchyme. It appears that cis-acting elements responsible for the complex retinoid regulation, tissue- and apoptosis-specific expression are embedded within the proximal 3.8 kb of DNA flanking the 5'-end of the mouse tissue transglutaminase gene.
Transglutaminases (Tg) are a family of calcium-dependent enzymes that catalyse the crosslinking of polypeptide chains, including those of extracellular matrix (ECM) proteins, through the formation of e(v-glutamyl) lysine bonds. This leads to the formation of protein polymers that are highly resistant to degradation. Consequently, tissue Tg (tTg) has been associated with ECM protein deposition in fibrotic diseases such as pulmonary fibrosis and atherosclerosis. hi this study, we have investigated the involvement of tTg in the development of kidney fibrosis in adult male Wistar rats submitted to subtotal nephrectomy (SNx). Groups of 4 to 6 rats were sacrified on days 7, 30, 90, and 120 after SNx. As previously described these rats developed progressive glomerulosclerosis and tubulo-interstitial fibrosis. The tissue level of e(y-glutamyi) lysine crosslink (as determined by exhaustive proteolytic digestion followed by cation exchange HPLC) increased from 347 + 94 pmol/mg protein (M.+S.E.M.) in controls to 1324 + 143 pmol/mg 90 days after SNx (p<0.01). Levels of crosslink correlated well with the renal fibrosis score throughout the 120 observation days (r=0.78, pO.Ol). Tissue homogenates showed no significant change in overall tTg activity (14C-putrescine incorporation assay) during this period. Immunohistochemistry demonstrated that this large increase in crosslink was predominantly in the cytoplasm of tubular cells and that these cells also showed a large increase in tTg antigen. Some of the cells high in crosslink and tTg displayed the morphology of a cell undergoing apoptosis, although these cells did not frequently show DNA cleavage (in-situ end labelling) typically associated with apoptosis. We noted an association between tTg, crosslink and renal scarring in rats submitted to SNx. We would like to postulate a role for tissue transglutamrnase in the development of experimental renal fibrosis.
Scarring and impaired wound repair are major causes of numerous clinical problems. In children the generation of scar tissue can interfere with normal growth and result in various deformities (such as those resulting from cleft lip and palate surgery or severe burns to the limbs and body). Scar tissue also impairs the function of many organs leading to disease processes such as liver cirrhosis, post transplant scarring of kidneys, lungs etc. as well as contributing to the generation of intestinal obstructions, adhesions and strictures, impaired vision and defective hearing. Impaired wound healing on the other hand results in chronic difficulties in treating conditions such as venous ulcers, diabetic ulcers and pressure sores. Finally, scarring often results in undesirable cosmetic abnormalities that have severe and significant psychological consequences.
Celiac disease is a complex autoimmune disease which is characterized by a strong genetic association (HLA-DQ2 or -DQ8), gluten as nutritional etiological factor, and the enzyme tissue transglutaminase as endomysial autoantigen. Patients develop highly predictive IgA autoantibodies to tTG. Certain gluten peptides are presented by the disease-associated HLA-DQ2/DQ8 molecules leading to stimulation of gluten-specific T cells. This immune response which is driven in the lamina propria causes the mucosal transformation characteristic for celiac disease. Increased intestinal expression of tTG in patients with CD appears to play an important role in the pathogenesis of CD. Thus, modification of gluten peptides by tTG, especially deamidation of certain glutamine residues, can enhance their binding to HLA-DQ2 or -DQ8 and potentiate T cell stimulation. Furthermore, tTG-catalyzed cross-linking and consequent haptenization of gluten with extracellular matrix proteins allows for storage and extended availability of gluten in the mucosa. New therapeutic approaches aim at proteolytic destruction of im-munodominant gliadin peptides that are resistant to intestinal enzymes by bacterial prolyl endopeptidases, the inhibition of tTG activity with highly specific enzyme inhibitors or at HLA-DQ2/DQ8 blocking peptide analogues.
Transglutaminase 2 (TG2) is a multifunctional protein that contributes to inflammatory disease when aberrantly expressed. Although macrophages express TG2, the factor stimulating TG2 expression remains poorly characterized in these cells. In the present study, we examined the effects of the stress-related catecholamines adrenaline and noradrenaline on macrophage expression of TG2 in RAW264.7 murine macrophages and murine bone marrow-derived macrophages. Treatment with adrenaline markedly increased TG2 mRNA expression and increased TG2 protein levels. While the β2-adrenoceptor-selective antagonist ICI 118,551 completely blocked adrenaline-induced TG2 mRNA expression, the β2-adrenoceptor specific agonist salmeterol increased TG2 expression. Noradrenaline also increased TG2 mRNA expression at higher doses than the effective doses of adrenaline. The effect of adrenaline on TG2 mRNA expression was mimicked by treatment with the membrane-permeable cAMP analog 8-Br-cAMP. Thus, increased intracellular cAMP following stimulation of β2-adrenoceptors appeared to be responsible for adrenaline-induced TG2 expression. Because stress events activate the sympathetic nervous system and result in secretion of the catecholamines, adrenoceptor-mediated increase in macrophage TG2 expression might be associated with stress-related inflammatory disorders.
Tissue transglutaminase is a calcium-dependent, protein cross-linking enzyme that is highly expressed in cells undergoing
apoptosis. The expression of tissue transglutaminase is regulated by a variety of molecules including retinoids, interleukin-6,
and transforming growth factor-β1 (TGF-β1). Retinoid and interleukin-6 inductions of tissue transglutaminase expression are
mediated by specificcis-regulatory elements located within the first 4.0 kilobase pairs of the promoter of the gene. The present studies were designed
to identify the molecular mechanisms mediating the regulation of tissue transglutaminase gene expression by TGF-β family members.
Transient transfection of Mv1Lu cells with transglutaminase promoter constructs demonstrated that 0.2 nm TGF-β1 maximally induced the activation of the promoter through a 10-base pair TGF-β1 response element (TRE; GAGTTGGTGC)
located 868 base pairs upstream of the transcription start site. This same element mediated an inhibitory activity of TGF-β1
on the transglutaminase promoter in MC3T3 E1 cells. The TRE through which TGF-β1-regulated the activity of the transglutaminase
promoter was necessary and sufficient for bone morphogenetic protein 2- (BMP) and BMP4-dependent inhibition of the tissue
transglutaminase promoter. The TGF-β1, BMP2, and BMP4 regulation of the transglutaminase promoter activity was similar to
the responses we observed for the endogenous transglutaminase activity of Mv1Lu and MC3T3 E1 cells. For BMP2 and BMP4, this
regulation was paralleled by a decrease in tissue transglutaminase mRNA in MC3T3 E1 cells. The results of these experiments
suggest that TGF-β1, BMP2, and BMP4 regulation of mouse tissue transglutaminase gene expression requires a composite TRE located
in the 5′-flanking DNA.
Epidermal transglutaminase-K is believed to catalyze the covalent linking of loricrin and involucrin to form cross-linked (CE) envelopes. In normal skin, transglutaminase-K is expressed as a band immediately below the stratum corneum, whereas in psoriasis and healing skin its expression is considerably expanded throughout the suprabasal layers. We have investigated whether the hyperproliferative state induced by short-term application of topical retinoic acid is similarly characterized by an increase in transglutaminase-K enzyme activity and immunoreactivity.
Retinoic acid (0.1% cream) or vehicle were applied to human skin and occluded for 4 d. Skin biopsies were obtained for measurement of transglutaminase-K and transglutaminase-C activity and immunoreactivity. For comparison, cultured normal human keratinocytes were incubated for 4 d in the presence of 1 μM retinoic acid and the subsequent transglutaminase-K activity and immunoreactivity measured. Transglutaminase-K activity was increased 2.8 times in retinoic acid compared to vehicle-treated skin (p < 0.005, n = 12) whereas there was no significant difference in transglutaminase-C activity. However, transglutaminase-K mRNA levels were not significantly different between retinoic acid- and vehicle-treated skin. In vehicle-treated skin, transglutaminase-K immunoreactivity was limited to a narrow, substratum corneal band, but was considerably expanded in a diffuse suprabasal pattern in retinoic acid-treated epidermis. In contrast, transglutaminase-K immunostaining was decreased and its enzymatic activity reduced sixfold in retinoic acid-treated keratinocytes (p < 0.01, n = 4).
These results demonstrate that retinoic acid treatment in vivo, in contrast to in vitro, leads to not only increased transglutaminase-K protein expression but also increased enzymatic activity in the absence of detectable increases in mRNA levels.These data, taken with the previously reported lack of in vivo modulation of the differentiation markers keratins 1 and 10 by retinoic acid, indicate that certain aspects of keratinocyte terminal differentiation that are altered in vitro by retinoic acid do not occur in vivo in human skin.
Lipases are highly active in the polymerization of a range of monomers. Both ring-opening polymerization of cyclic monomers such as lactones and carbonates as well as polycondensation reactions have been investigated in great detail. Moreover, in combination with other (chemical) polymerization techniques, lipase-catalyzed polymerization has been employed to synthesize a variety of polymer materials. Major advantages of enzymatic catalysts are the often-observed excellent regio-, chemo- and enantioselectivity that allows for the direct preparation of functional materials. In particular, the application of techniques such as Dynamic Kinetic Resolution (DKR) in the lipase-catalyzed polymerization of racemic monomers is a new development in enzymatic polymerization. This paper reviews selected examples of the application of lipases in polymer chemistry covering the synthesis of linear polymers, chemoenzymatic polymerization and applications of enantioselective techniques for the synthesis and modification of polymers.
This review summarises the functions of the enzyme tissue transglutaminase (TG2) in the extracellular matrix (ECM) both as a matrix stabiliser through its protein cross-linking activity and as an important cell adhesion protein involved in cell survival. The contribution of extracellular TG2 to the pathology of important diseases such as cancer and fibrosis are discussed with a view to the potential importance of TG2 as a therapeutic target. The medical applications of TG2 are further expanded by detailing the use of transglutaminase cross-linking in the development of novel biocompatible biomaterials for use in soft and hard tissue repair.
Three days after biopsy wounds were made in the dorsal skin of rats the animals were killed and explants of wounded and unwounded skin were incubated for 7 h with either [3H]glutamine or [3H]lysine. Both incubated and fresh control explants were then dissected into three layers which were homogenized, extracted, digested and then assayed for epsilon (gamma-glutamyl)lysine. The concentration of epsilon(gamma-glutamyl)lysine was greater in all three wounded layers than in the corresponding unwounded layers. The concentration in the wounded middle (dermal) layer and in the unwounded middle layer of younger rats was greater than in the unwounded outer (keratinized) layer, which has previously been shown to contain epsilon(gamma-glutamyl)lysine crosslinks. The incorporation of label from both [3H]glutamine and [3H]lysine into buffer-insoluble protein of the middle and inner (muscle) layers was much greater in the wounded explants than in the unwounded. Except for [3H]lysine in the inner layer there was also an increase in the fraction of incorporated label which was converted to epsilon(gamma-glutamyl)lysine. These results show that increased protein biosynthesis during repair in the wounded explants is associated with increased formation of epsilon(gamma-glutamyl)lysine. In addition, they indicate that the crosslink is involved in some process in the middle and inner layers which is distinct from its known function in keratinization of the epidermis.
Osteopontin (OPN), an extracellular matrix cell adhesion protein, was found to serve as a substrate for the incorporation of radiolabelled putrescine mediated by a commercial preparation of guinea pig liver transglutaminase. Preliminary evidence also suggests that OPN serves as a substrate for the plasma transglutaminase, Factor XIIIa. While the protein substrates to which OPN is linked in vivo have not been identified, it is reasonable to speculate that this capacity of OPN may dictate its extracellular location and thereby affect its role in bone homeostasis, tumorigenesis, metastasis, resistance to bacterial infections or, perhaps, wound repair.
By use of immunoperoxidase staining, tissue transglutaminase and aminopropeptide of collagen type III were localized to fibroblasts running within cords of Dupuytren's fascia. Quantitative analysis of transglutaminase revealed that activity levels were significantly higher in acutely contracting fascia than in chronic contractures. The results show that contractures in Dupuytren's fascia may be reflected by the level of transglutaminase activity in the tissue. Furthermore, it is suggested that isopeptide bond formation, involving collagen type III aminopropeptide moieties, may be the biochemical mechanism by which transglutaminase maintains the contracted state.
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.
Rabbits were fed for 10-12 weeks on a normal pellet diet or on the same diet containing 1% cholesterol and 6% peanut oil. The animals were killed and the aortas divided into three layers which were homogenized and extracted. The extracts and the insoluble residues were assayed for transglutaminase activity and tissue transglutaminase antigen. When compared with normal aortas, the inner and middle layers of aortas with atherosclerotic lesions from cholesterol-fed rabbits showed higher transglutaminase activities in the buffer-soluble fraction without a corresponding increase in antigen. The buffer extracts showed two peaks (I and II) of activity and antigen on DE 52 chromatography; peak I was also found, together with lipid, in Triton X-100 extracts of the buffer-insoluble residue. The Triton X-100 insoluble fraction showed higher concentrations of both activity and antigen in the inner and middle layers of atherosclerotic aortas than in normal aortas, but the activity per nanogram of antigen was lower than in the buffer-soluble fraction. The activity in this insoluble residue was largely extracted, together with an inhibitor, by an NaCl-sucrose-dithiothreitol-Triton X-100 solution. DE 52 chromatography of this extract showed a third peak of activity and antigen (peak III) and an inhibitor peak that was distinct from the activity peaks.
Factor XIII Human epidermis
The problem of the biochemical quantification of long term human wound healing was approached by measuring collagen synthesis in reincisions using specific radioimmunoassays for the wound fluid concentrations of the carboxyterminal propeptide of type I procollagen (PICP) and the aminoterminal propeptide of type III procollagen (PIIINP). First-day wound fluid PICP concentration after reincising a 3 week old scar was 25 times higher than the mean value in 20 reference standard incisions but scars older than 3 months did not show this difference. Wound fluid taken in subsequent days demonstrated that the initial acceleration of synthesis disappeared by the fourth day. When wound fluid PIIINP was assessed, high concentrations were found in reincisions of wounds for up to 5 months after the previous operation. The acceleration was also lost more slowly during the first postoperative week. The duration of a high rate of type I collagen synthesis compares well with studies in experimental wounds which show increased gain of strength if they are made not more than 6 weeks after previous surgery. The longer activity of the metabolism of type III collagen related antigens could reflect their function in the regulation of collagen fibril formation.
Tissue type II transglutaminase (TGase) is a member of the TGase family that catalyzes Ca(2+)-dependent covalent cross-linking of several amines to the gamma-carboxamide group of protein-bound glutamine residues. The degree of therapeutic efficacy or toxicity of drugs may be related to their ability to serve as a substrate for TGase and their covalent linkage to glutamine residues of regulatory proteins through the catalytic action of this enzyme. Here, doxorubicin (adriamycin)-resistant human breast carcinoma MCF-7ADR cells exhibited 40- to 6C-fold higher TGase activity than control drug-sensitive MCF-7WT cells. The same was observed in vivo: a small proportion of tumor cells became positive for TGase after administration of adriamycin-based chemotherapy to patients with breast carcinoma. Similarly, continuous culture of MCF-7WT cells in the presence of adriamycin led to the appearance of the drug-resistant phenotype that was in turn associated with increased expression of TGase. This increase in TGase was specific for adriamycin resistance. Like most known TGase, MCF-7ADR TGase was completely dependent on the presence of Ca2+ for its catalytic activity. Based on its immunoreactivity, the TGase in MCF-7ADR cells was identified as an 85-kDa tissue-type TGase and was present only in the soluble form. Immunoblot analysis revealed that the increase in TGase activity was due to accumulation of the protein. Two cytosolic proteins of approximately 20 and 30 kDa in MCF-7 cells served as suitable acyl donor substrates in TGase-catalyzed reactions.
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), has diverse effects in a variety of tissues and cell types, including skin. Since 1,25(OH)2D3 affects both fibroblast and keratinocytes, we evaluated the effect of 1,25(OH)2D3 on wound healing. We investigated the effect of the topically applied 1,25(OH)2D3 or vehicle on the healing of cutaneous wounds in rats in a blinded manner. Wound areas were measured by planimetry technique. Healing was expressed as the percentage of the original wound area that was healed. 1,25(OH)2D3 at concentrations between 5 and 50 ng/day caused a dose-dependent acceleration of healing. Time course and specificity studies indicated that 1,25(OH)2D3 specifically promoted healing between 1-5 days after wounding as compared with vitamin D (0.5 microgram/day), which showed no significant improvement over control. Our results suggest that 1,25(OH)2D3 and its analogues may be a new class of compounds that could be developed to enhance wound healing.
A 2.4 kilobase (kb) cDNA encoding a new form of human tissue transglutaminase homologue (TGH2) was isolated from retinoic acid-induced human erythroleukemia cell (HEL) library. Full-length cDNA analysis gives an open reading frame coding for a polypeptide of 349 amino acid residues with a molecular mass of 38,700 Da. This variant differs from the previously reported homologue TGH in that it is 199 amino acids shorter and has an alternative, 63 amino acid COOH-terminal peptide. The 3'-untranslated region of the cDNA also differs from the previously reported sequences for both TGH and human tissue transglutaminase. The region coding for the first 286 amino acids of TGH2, which contains the active site is identical to TGH. Immunoprecipitation of the in vitro translation product from a synthetic TGH2 mRNA and immunoprecipitation of total protein of human heart, liver, kidney and cultured erythroleukemia HEL cell, revealed a protein with a molecular mass of 37,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the cDNA sequence for the previously known tissue transglutaminases with genomic DNA and the TGH2 cDNA described here indicate that the sequence divergence points correlate with known intron-exon boundaries. The smaller RNA species encode for truncated proteins with novel carboxyl termini. The TGH cDNA and the TGH2 cDNA both produce transcripts which start with the regular coding sequence for TGase and then fail to splice at specific donor sites, resulting in the use of an alternative exon that contains a stop codon.
Tissue transglutaminase (transglutaminase type II) is an intracellular protein cross-linking enzyme that accumulates in connective tissue and in cells undergoing apoptosis. Retinoids regulate the transcription of the mouse tissue transglutaminase gene via activation of regulatory elements contained within 4 kilobases of the 5'-end of the gene. Co-transfection studies with retinoid receptor expression vectors in CV-1 cells demonstrated that the mouse tissue transglutaminase promoter is activated by ligand activation of either retinoic acid receptor-retinoid X receptor (RAR.RXR) heterodimers or RXR homodimers. Optimal induction is achieved with retinoid receptor panagonists; partial activation can also be achieved with either RAR-specific or RXR-specific retinoids. Retinoid-dependent activation of the tissue transglutaminase promoter depends on both a proximal regulatory region containing sequences highly conserved between the human and the mouse tissue transglutaminase promoters and a distal region that includes a 30-base pair retinoid response element (mTGRRE1). mTGRRE1 contains three hexanucleotide half-sites (two canonical and one non-canonical) in a DR7/DR5 motif that bind both RAR*RXR heterodimers and RXR homodimers. These studies suggest that retinoid-dependent expression of the mouse tissue transglutaminase gene is mediated by a versatile tripartite retinoid response element located 1.7 kilobases upstream of the transcription start site.
Transglutaminase (TGase) activities were measured in rat tissues 1-7 days after intraperitoneal injection of saline or lipopolysaccharide (LPS) and in the cells and media from pre-confluent human fibroblasts cultured for two days in the presence or absence of LPS. epsilon (gamma-Glutamyl)lysine and [3H]putrescine-labelled gamma-glutamyl derivatives in extracellular and cellular fibroblast proteins were also measured. Three effects of LPS were observed. Firstly, total TGase activity is greater in the tissues from the LPS-injected animals, with the maximum increase occurring at 1 day in dermis, epidermis and liver, at 5 days in the aorta and, after a decrease at 2-5 days, at 7 days in the panniculus muscle. Secondly, the fraction of the total activity which is buffer-extractable is greater on days 1 and/or 2 in all the tissues from the LPS-injected rats. Thirdly, in cultures of human fibroblasts, LPS increases that fraction of bound [3H]putrescine and of TGase and its gamma-glutamylamine products which occurs in the extracellular medium. In addition, a higher concentration of TGase-derived crosslinks was found in extracellular as opposed to intracellular proteins. In conjunction with previous findings in skin wound healing and in atherosclerosis these results support the concept of an extracellular function for tissue TGase and indicate that there is a widespread association of increases in TGase and its extracellular products with inflammation and the healing or fibrotic processes which follow it.
Transglutaminase (TGase) is a calcium-dependent enzyme which catalyzes the iso-peptide cross-link between peptide-bound glutamine and lysine in vivo. Though the cross-link is developed as a barrier function in the skin system, overexpression of this could invoke skin hyperkeratosis in psoriasis and roughness in aged skin. In former research, many strong irreversible TGase inhibitors failed application because of high cytotoxicity. We selected one peptide after primary screening of six synthetic peptides designed from domains of known TGase substrates. Then we attempted to reduce the size and finally obtained two tetrameric peptides. When we treated keratinocyte with these TGase inhibitors under calcium-induced differentiation, the formation of a cornified cell envelope (CE) was decreased to the same level of CE under proliferating conditions without cytotoxic effect. Therefore, we propose that these TGase inhibitors may be useful for solving the physiological hypercross-linking problems for pharmaceutical or cosmetic purposes.
Tissue transglutaminase is a calcium-dependent enzyme that catalyzes the cross-linking of polypeptide chains, including those of extracellular matrix (ECM) proteins, through the formation of epsilon-(gamma-glutamyl) lysine bonds. This crosslinking leads to the formation of protein polymers that are highly resistant to degradation. As a consequence, the enzyme has been implicated in the deposition of ECM protein in fibrotic diseases such as pulmonary fibrosis and atherosclerosis. In this study, we have investigated the involvement of tissue transglutaminase in the development of kidney fibrosis in adult male Wistar rats submitted to subtotal nephrectomy (SNx). Groups of six rats were killed on days 7, 30, 90, and 120 after SNx. As previously described, these rats developed progressive glomerulosclerosis and tubulo-interstitial fibrosis. The tissue level of epsilon-(gamma-glutamyl) lysine cross-link (as determined by exhaustive proteolytic digestion followed by cation exchange chromatography) increased from 3.47+/- 0.94 (mean+/-SEM) in controls to 13.24+/-1.43 nmol/g protein 90 d after SNx, P
The cross-linking enzyme tissue transglutaminase (tTG) participates in a variety of cellular functions. To assess its contribution to extracellular and intracellular processes during development we cloned the cDNA for chicken heart tissue transglutaminase and localized the sites of transglutaminase expression by in situ hybridization and immunohistochemistry. Compared with the chicken red blood cell transglutaminase cDNA, the heart cDNA encodes a transglutaminase with an amino-terminal truncation. The truncated enzyme retains full catalytic activity and is GTP-inhibitable. Tissue transglutaminase expression was observed in developmentally transient structures in embryonic chicken limb at day 7.5 of incubation suggesting that its expression is dynamically regulated during limb morphogenesis. The major morphogenetic events of the limb associated with transglutaminase expression were cartilage maturation during skeletal development, interdigital apoptosis, and differentiation of skeletal muscle. Maturation of the cartilage during endochondral ossification was characterized by intra- and extracellular transglutaminase accumulation in the zone of hypertrophic chondrocytes. Only intracellular enzyme could be detected in mesenchymal cells of the prospective joints, in apoptotic cells of the interdigital web, and in skeletal muscle myoblasts. An apparently constitutive expression of tissue transglutaminase was found in vascular endothelial cells corresponding to the adult expression pattern. The dynamic pattern of transglutaminase expression during morphogenesis suggests that tissue remodeling is a major trigger for transglutaminase induction.
It was recently demonstrated that renal tissue transglutaminase (tTg) protein and its catalytic product the epsilon(gamma-glutamyl) lysine protein cross-link are significantly increased in the subtotal (5/6) nephrectomy model (SNx) of renal fibrosis in rats. It was proposed that the enzyme had two important physiologic functions in disease development; one of stabilizing the increased extracellular matrix (ECM) by protein cross-linking, the other in a novel form of tubular cell death. This study, using the same rat SNx model, demonstrates first by Northern blotting that expression of tTg mRNA when compared with controls is increased by day 15 (+70% increase, P < 0.05), then rises steadily, peaking at day 90 (+391%, P < 0.01), and remains elevated at 120 d (+205%, P < 0.05) when compared with controls. In situ hybridization histochemistry demonstrated that the tubular cells were the major site of the additional tTg synthesis. Immunohistochemistry on cryostat sections revealed a sixfold increase (P < 0.001) in ECM-bound tTg antigen at 90-d post-SNx, whereas in situ transglutaminase activity demonstrated by the incorporation of fluorescein cadaverine into cryostat sections indicated a 750% increase (P < 0.001) on day 90 in SNx animals. This increased activity was extracellular and predominantly found in the peritubular region. These results indicate that increased tTg gene transcription by tubular cells underlies the major changes in renal tTg protein reported previously in SNx rats, and that the presence of the epsilon(gamma-glutamyl) lysine cross-links in the extracellular environment is the result of the extracellular action of tTg. These changes may be in response to tubular cell injury during the scarring process and are likely to contribute to the progressive expansion of the ECM in renal fibrosis.
Tissue transglutaminase (TG) is an enzyme that stabilizes the structure of tissues by covalently ligating extracellular matrix molecules. Expression and localization of TG are not well established during wound healing. We performed punch biopsy wounds on anesthetized rats and monitored the wound healing process by histological and immunohistochemical methods. The TG antigen and activity are expressed at sites of neovascularization in the provisional fibrin matrix within 24 h of wounding. Endothelial cells, macrophages, and skeletal muscle cells expressed TG throughout the healing process. The TG antigen within the wound was active in vivo based on the detection of isopeptide bonds. The TG antigen increased four- to fivefold by day 3 postwounding and was proteolytically degraded. TG expression occurred in association with TGF-beta, TNF-alpha, IL-6, and VEGF production in the wound. Recombinant TG increased vessel length density (a measure of angiogenesis) when applied topically in rat dorsal skin flap window chambers. We have established that TG is an important tissue stabilizing enzyme that is active during wound healing and can function to promote angiogenesis.
The calcium-dependent cross-linking enzyme tissue transglutaminase (tTgase, type II) is a potential novel player at the cell surface, where its contribution to cell adhesion and stabilization of the extracellular matrix is becoming increasingly recognized. We investigated whether tTgase enhances the biological recognition of poly (DL lactide co-glycolide) (PLG), poly (epsilon-caprolactone) (PCL), and poly (L lactide) (PLA), biomaterials widely used in medical implants. Three cell-model systems consisting of human osteoblasts, endothelial cells (ECV-304), and Swiss 3T3 fibroblasts were utilized, in which tTgase expression was modulated by gene transfer, and the ability of cells to spread on these polymers was quantified in relation to the altered level of expressed tTGase. Results show that over-expression of tTgase in human osteoblasts positively correlated with cell spreading on PLG, while no attachment and spreading was found on PCL and PLA. Antisense silencing of tTgase in the endothelial cells led to a marked reduction of cell spreading on all polymers. The hydrophobic nature of PLC also appeared to favor endothelial cell attachment. Spreading of Swiss 3T3 fibroblasts on these biomaterials was only slightly affected by increased expression of tTgase, although cell spreading on control glass was increased. We propose that the consideration of tTgase-mediated bioactivity in novel biomaterials may improve cell attachment and promote biocompatibility.
Transglutaminase 1 (TGase 1) is a Ca(2+)-dependent enzyme which catalyzes epsilon-(gamma-glutamyl)lysine cross-linking of substrate proteins such as involucrin and loricrin to generate the cornified envelope at the cell periphery of the stratum corneum. We have shown that disruption of the TGase 1 gene in mice results in neonatal lethality, absence of the cornified envelope, and impaired skin barrier function. Based on the importance of TGase 1 in epidermal morphogenesis, we have now assessed its role in wound healing. In neonatal mouse skin, TGase 1 mRNA as well as keratin 6alpha was induced in the epidermis at the wound edges as early as 2 hours after injury and that expression continued in the migrating epidermis until completion of re-epithelialization. The TGase 1 enzyme co-localized on the plasma membrane of migrating keratinocytes with involucrin, but not with loricrin, which suggests the premature assembly of the cornified envelope. Similar injuries to TGase 1 knockout mouse skins grafted on athymic nude mice showed substantial delays in wound healing concomitant with sustained K6alpha mRNA induction. From these results, we suggest that activation of the TGase 1gene is essential for facilitated repair of skin injury.
To determine the expression and distribution of tissue transglutaminase (TG(C)) and extracellular matrix (ECM) proteins in rat cornea during epithelial wound healing.
Corneal epithelial defects were created in rat corneas, and TG(C) expression was examined by Northern blot analysis, in situ hybridization, and immunohistochemical staining after the injury. The presence of fibrinogen, laminin-1, nidogen/entactin, and type collagen was also determined immunohistochemically.
TG(C) was expressed in normal corneas. During the early wound healing process, TG(C) mRNA expression was up-regulated and TG(C) immunoreactivity was predominantly expressed in the migrating epithelial cells. ECM proteins were also expressed in a similar pattern as TG(C).
The sites and time course of TG(C) expression indicate that TG(C) probably plays a role in maintaining the homeostasis of the cornea and in promoting epithelial wound healing. The simultaneous expression of TG(C) and ECM proteins suggests that the ECM proteins probably operate in concert with TG(C) in corneal wound healing.
SACCHACHITIN is a skin wound-healing membrane made of residual fruiting body of Ganoderma tsugae. Its effect on proliferating cell nuclear antigen (PCNA) expression in actively proliferating cells, type I collagen expression and tissue remodeling in the healing tissue, and the association of tissue-transglutaminase (t-TGase) with wound healing were investigated by immunohistochemical staining. The results demonstrated that PCNA expressed in keratinocytes since day 1 in the SACCHACHITIN group and persisted during entire healing process. In contrast, it was barely detectable on day 3 in the control group. At keratinocyte layer, the SACCHACHITIN group exhibited more type I collagen than did the control group since day 1. At scar tissue, type I collagen was positively stained in the SACCHACHITIN group since day 7 but not in the control group till day 12. Furthermore, t-TGase was strongly expressed on the inner wall of angiogenic vessels on day 5 of the control group but not on that of the SACCHACHITIN group until day 10. The earlier expressions of PCNA and type I collagen in the keratinocyte layer may lead to accelerated skin wound healing. In addition, the later expression of t-TGase, an indicator of apoptosis, on the inner wall of angiogenic capillaries in the SACCHACHITIN group may indicate a longer period of blood supply to the wound area, thus facilitating wound healing. These observed phenomena might underline the beneficial effects of SACCHACHITIN membrane on rapid wound healing.
A complex series of events involving inflammation, cell migration and proliferation, ECM stabilisation and remodelling, neovascularisation and apoptosis are crucial to the tissue response to injury. Wound healing involves the dynamic interactions of multiple cells types with components of the extracellular matrix (ECM) and growth factors. Impaired wound healing as a consequence of aging, injury or disease may lead to serious disabilities and poor quality of life. Abnormal wound healing may also lead to inflammatory and fibrotic conditions (such as renal and pulmonary fibrosis). Therefore identification of the molecular events underlying wound repair is essential to develop new effective treatments in support to patients and the wound care sector.
Recent advances in the understating of the physiological functions of tissue transglutaminase a multi functional protein cross-linking enzyme which stabilises tissues have demonstrated that its biological activities interrelate with wound healing phases at multiple levels. This review describes our view of the function of tissue transglutaminase in wound repair under normal and pathological situations and highlights its potential as a strategic therapeutic target in the development of new treatments to improve wound healing and prevent scarring.
The aminopropeptide of type III collagen incorporates [3H]putrescine in the presence of liver transglutaminase, and the change in incorporation with concentration indicates one binding site on each of the Mr = 15,000 subunits of the peptide. At low concentrations the incorporation was comparable to that of dimethyl casein and much greater than actin or fibrinogen. Cleavage and Edman degradation of the aminopropeptide identified the major putrescine-binding site as glutamine in position 14. The surrounding amino acid sequence (Leu-Gly-Gln-Ser) shows homology with some synthetic peptide substrates of transglutaminase.
An experimental model of pulmonary fibrosis has been developed by dosing rats with one-fifth the LD50 dose of the herbicide paraquat on 5 consecutive days. Approximately 50% of the rats died within 4 days of the completion of dosing, showing macroscopic changes and wet weight increases in the lung consistent with severe oedema. Those animals which died between Days 4 and 10 had markedly increased levels of hydroxyproline in the lung, maximum at Day 6, and increased prolyl hydroxylase activity, maximum at Day 4. These changes, together with an increase in thymidine incorporation into DNA, and increased lung DNA content, were consistent with the development of fibrosis. Measurement of transglutaminase activity in the lung showed marked increases at Days 4 and 10 after completion of dosing. This activity paralleled closely the changes in prolyl hydroxylase activity and became increasingly associated with particulate protein present in the "nuclear pellet" fraction. The presence of zymogen plasma transglutaminase trapped in lung homogenates could not be demonstrated but the contribution by the active plasma transglutaminase (Factor XIIIa) to increases shown at Day 4 cannot be ruled out.
Addition of retinoic acid to human promyelocytic leukemia cells results in a dramatic increase in cellular transglutaminase activity. This increase is due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. Retinoic acid-induced expression of tissue transglutaminase is potentiated by analogues of cyclic AMP. The induction of the enzyme can be detected within 6 h of the addition of the retinoid to the cell and results in increases of the enzyme of at least 50-fold. The induction of HL-60 transglutaminase is a specific response of the cells to retinoic acid and is not seen with other agents that induce HL-60 differentiation. We believe that the induction of tissue transglutaminase is a useful index of the early events in retinoid-regulated gene expression in both normal and transformed cells.
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.
Transglutaminases are a class of enzymes capable of covalently cross-linking both intracellular and extracellular proteins. The activity of tissue transglutaminase is known to decrease precipitously following neoplastic transformation, and it has been hypothesized that transglutaminase may be involved in growth regulation. We have found that the differentiation promoter sodium butyrate is able to cause a marked increase in transglutaminase activity in PC12 pheochromocytoma cells in a time- and dose-dependent manner. This increased transglutaminase activity is associated with growth arrest, as well as with striking morphological changes including increased cell adhesion. The transglutaminase induced by sodium butyrate appears to be tissue transglutaminase, based on its cytosolic localization, thermal lability at basic pH, and elution profile on anion-exchange chromatography. Untreated PC12 cells contain only small amounts of transglutaminase which resembles epidermal transglutaminase, an enzyme previously described only in skin. In contrast to sodium butyrate, nerve growth factor did not stimulate tissue transglutaminase in PC12 cells, although it, too, caused growth arrest. It is hypothesized that transglutaminase may be involved in certain morphological changes accompanying cellular differentiation and neoplastic transformation, rather than in growth regulation per se.
Evidence is presented that transglutaminase is composed of a single polypeptide chain of molecular weight 80,000 to 90,000. (a) Polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and mercaptoethanol gave a single band with a mobility corresponding to a molecular weight of approximately 85,000. (b) Gel filtration in guanidine HCl of the ¹⁴C-carbamidomethyl carboxymethylated enzyme protein showed a single peak of absorbance and radioactivity from which a molecular weight of approximately 85,000 was estimated. (c) Amino-terminal analysis by conventional methods showed no evidence of free α-amino groups. A peptide, believed to contain the amino-terminal residue, was obtained by Pronase digestion and was isolated at levels of 0.75 and 0.8 mole/90,000 g of enzyme. The sequence of this peptide was determined as pyroglutamylalanylaspartylleucine. (d) Digestion by carboxypeptidase A of the carboxymethylated enzyme protein in denaturing solvents released glycine and serine at equal rates to the level of 1 mole/90,000 g of protein. Hydrazinolysis gave approximately 1 mole of glycine. These findings, together with earlier evidence that the molecular weight of the native enzyme is 80,000 to 90,000 and that the enzyme protein contains 17 or 18 —SH groups, but no disulfide bonds, form the basis for the view of an unbridged monomeric structure of transglutaminase.
Indication that transglutaminase performs its catalytic functions in the monomeric form was obtained from a comparison of the gel filtration patterns for the enzyme protein in the presence and absence of calcium ion. The identical nature of these patterns is in accord with the suggestion that this metal, which is essential for activation of transglutaminase, does not affect a change in enzyme molecular weight.
A revised enzyme purification procedure is presented. Rabbit antiserum against transglutaminase has been prepared and used to characterize the enzyme purified by this procedure as immunologically homogeneous.
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.
Transglutaminase (R-glutaminyl-peptide:amine alpha-glutamyl-yltransferase [EC 2.3.2.13]) has been purified to apparent homogeneity from extracts of rabbit liver. The enzyme is a single polypeptide chain of approximately 80 000 molecular weight containing one catalytic site per molecule. That the isolated enzyme is the rabbit counterpart of the well-characterized guinea pig liver transglutaminase is evidenced by the similarities in their amino acid compositions and in their enzymic activities toward several substrates, together with the fact that the isolated rabbit enzyme is immunologically distinct from both rabbit plasma and rabbit platelet blood coagulation factor XIII. A striking difference between the catalytic activities of the rabbit and guinea pig enzymes is the low activity of rabbit transglutaminase for hydroxylamine incorporation into benzyloxycarbonyl-L-glutaminylglycine, a reaction for which the guinea pig enzyme shows a high reactivity. This finding reveals the cause of error in an earlier report (Tyler, H.M., and Laki, K. (1967) Biochemistry 6, 3259) that rabbit liver contains little, if any, of the enzyme. Preparation of, and analytical data on, several glutamine-containing peptide derivatives used in this study are reported here.
The predominant form of the cross-linking enzyme, transglutaminase, in cultured normal human epidermal keratinocytes, is found in cell particulate material and can be solubilized by nonionic detergent. It elutes as a single peak upon either anion-exchange or gel-filtration chromatography. Monoclonal antibodies raised to the particulate enzyme cross-react with one of two transglutaminases in the cell cytosol. The second cytosolic transglutaminase, which has distinct kinetic and physical properties from the first, does not cross-react and is not essential for formation of the keratinocyte cross-linked envelope in vitro. The anti-transglutaminase antibodies stain the more differentiated layers of epidermis in a pattern similar to that given by anti-involucrin antiserum. These observations support the hypothesis that the transglutaminase so identified is involved in cross-linked envelope formation in vivo.
A cellular enzyme-linked immunosorbent assay was developed for estimating cellular transglutaminase in situ using a monoclonal antibody produced to tissue transglutaminase. The minimum level of detection of TGase was 2-5 ng. The enzyme was present in greater amounts in WI-38 and IMR90 cells than in their simian virus-transformed counterparts. The levels of TGase in the virus-transformed cells increased significantly when the cells were grown in the presence of sodium butyrate to induce enzyme activity. Staining of confluent WI-38 cells by indirect immunofluorescence using the monoclonal antibody showed microscopic fibers suggesting that the enzyme may be associated with detergent-insoluble components.
The cellular distribution of active and inactive transglutaminase (TGase) was studied in C6 glioma cells before and during stimulation by a serum-containing medium. The activity of the enzyme was determined in the soluble and insoluble fractions obtained by freezing and thawing the cells, followed by centrifugation at 12,000g for 5 min. In the soluble fractions, the activity of TGase decreased 2.5 h post-stimulation and increased after 5 and 8 h. In the corresponding insoluble fractions, no significant changes in the activity of the enzyme were noted up to 8 h after stimulating the cells with fresh medium. An immunological approach was next used to determine the quantity of TGase antigen during the stimulation of the cultured glioma cells. In the soluble fraction, the quantity of the antigen decreases significantly at 2.5, 5, and 8 h. In contrast, in the insoluble fraction, a significant increase in TGase antigen was detected 8 h after the addition of fresh medium. Cycloheximide completely inhibited the increase in the quantity of TGase antigen in the insoluble fraction, 8 h post-stimulation, while actinomycin D caused a partial inhibition. Trypsin, neuraminidase, or Sendai viruses increased the activity of TGase significantly, when added to nonstimulated cells. Trypsin had no effect on TGase activity when added to the cells 2 h after stimulation with a serum-containing medium. These findings suggest that an inactive form of the enzyme is present in the insoluble cellular fraction. A model has been proposed to explain the variations in TGase activity, its distribution and translocation during cellular stimulation.
The aminopropeptide of type III collagen incorporates [3H]putrescine in the presence of liver transglutaminase, and the change in incorporation with concentration indicates one binding site on each of the Mr = 15,000 subunits of the peptide. At low concentrations the incorporation was comparable to that of dimethyl casein and much greater than actin or fibrinogen. Cleavage and Edman degradation of the aminopropeptide identified the major putrescine-binding site as glutamine in position 14. The surrounding amino acid sequence (Leu-Gly-Gln-Ser) shows homology with some synthetic peptide substrates of transglutaminase.
Cellular transglutaminase activity was induced in simian virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) by sodium butyrate. The level of enzyme activity approached a maximum by 6 days; 9-11-fold higher in the presence of sodium butyrate (1 mM) than in its absence. The observed increases in cellular transglutaminase activity could be entirely accounted for by equivalent increases in the levels of enzyme protein measured by inhibition enzyme-linked immunosorbent assay. Sodium butyrate also increased the rate of enzyme synthesis, but had no effect on the rate of cellular transglutaminase degradation. The increase in the rate of enzyme synthesis was matched by an increased level of translatable transglutaminase mRNA as measured in a cell-free translation system. Our results suggest that sodium butyrate regulates cellular transglutaminase at the pretranslational level.
To study the glycoproteins and transglutaminase substrates involved in healing, wounds were made in the skin of anesthetized rats with a biopsy punch. Explants made 1-5 days later were incubated with [3H]-labelled putrescine, fucose or proline. As compared with unwounded skin there was an increased incorporation of label which was greatest at 3 days. Incubation for various times suggests that the incorporation of fucose and proline is dependent on protein synthesis, whereas putrescine is incorporated into preformed proteins. Putrescine and fucose label polypeptides with an Mr of about 45,000 before and 14,000 after reduction. These correspond in size with the aminopropeptide of type III collagen. Other labelled material of higher molecular weight is partly degraded to similar polypeptides on collagenase digestion. Much of the [3H]putrescine in the polypeptides is in the form of gamma-glutamyl putrescine. It is hypothesized that isopeptide linkage of the aminopropeptide III occurs in wound healing.
Nondenaturing electrophoresis was used to demonstrate that, immediately upon exposure to plasma, the transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) from erythrocytes undergoes a significant shift in mobility. The plasma effect shows saturable characteristics and depends entirely on the presence of fibronectin in plasma, indicative of complex formation between this protein and transglutaminase. The results suggest a specific carrier function for fibronectin that might be of physiological importance in determining the fate of a tissue transglutaminase accidentally discharged into plasma.
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.
1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5'-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together.
This paper is intended as a background to the topic of transglutaminases, while focusing on current ideas regarding the biological roles of these enzymes. Specifically, the following topics are discussed: geometry of forming ?-glutamyl-e-lysine cross-linked structures; energetic considerations; the ?-glutamyl-e-lysine cross-link; amine incorporation assays; artefactual incorporation of amines in cells and tissue homogenates; synthetic substrate systems; regulation of transglutaminase activities; strategies for probing transglutaminase-mediated events in biological systems; the blood clotting paradigm; transglutaminase and cell aging: the Ca2+-enriched human erythrocyte; transglutaminase and cell activation: the thrombin-stimulated human platelet and the fertilized sea urchin egg.
This paper is intended as a background to the topic of transglutaminases, while focusing on current ideas regarding the biological roles of these enzymes. Specifically, the following topics are discussed: geometry of forming gamma-glutamyl-epsilon-lysine cross-linked structures; energetic considerations; the gamma-glutamyl-epsilon-lysine cross-link; amine incorporation assays; artefactual incorporation of amines in cells and tissue homogenates; synthetic substrate systems; regulation of transglutaminase activities; strategies for probing transglutaminase-mediated events in biological systems; the blood clotting paradigm; transglutaminase and cell aging: the Ca2+-enriched human erythrocyte; transglutaminase and cell activation: the thrombin-stimulated human platelet and the fertilized sea urchin egg.
The sequential appearance of fibronectin and collagenous proteins was studied by immunofluorescence in experimental granulation tissue in the mouse. Granulation tissue was induced by subcutaneous implantation of viscous cellulose sponges into the neck. When sponges were invaded by granulation tissue, fibronectin appeared early together with the invading fibroblasts. Interstitial collagens, type III and type I, appeared 2 to 7 days later. When the collagen had matured into bundles, fibronectin diminished or disappeared. The results suggest that fibronectin functions as a primary matrix for organization of the collagenous connective tissue during the tissue repair process.