RESUMEN
Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca(2+)-dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins and γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the K m and the V max kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild-type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2-driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of crosslinked proteins correlates with the manifestation of degenerative disorders.
Asunto(s)
Aminas/metabolismo , Liasas de Carbono-Nitrógeno/química , Liasas de Carbono-Nitrógeno/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Transglutaminasas/química , Transglutaminasas/metabolismo , Calcio/metabolismo , Liasas de Carbono-Nitrógeno/genética , Dominio Catalítico , Proteínas de Unión al GTP/genética , Humanos , Cinética , Mutación Missense , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/genéticaRESUMEN
Transglutaminases (TGs) stabilize proteins by the formation of ε(γ-glutamyl)lysine cross-links. Here, we demonstrate that the cross-linking of collagen I (COL I) by tissue transglutaminase (TG2) causes an alteration in the morphology and rheological properties of the collagen fibers. Human osteoblasts (HOB) attach, spread, proliferate, differentiate and mineralize more rapidly on this cross-linked matrix compared to native collagen. When seeded on cross-linked COL I, HOB are more resistant to the loss of cell spreading by incubation with RGD containing peptides and with α1, α2 and ß1 integrin blocking antibodies. Following adhesion on cross-linked collagen, HOB show increased phosphorylation of the focal adhesion kinase, and increased expression of ß1 and ß3 integrins. Addition of human bone morphogenetic protein to HOB seeded on TG2 cross-linked COL I enhanced the expression of the differentiation marker bone alkaline phosphatase when compared to cross-linked collagen alone. In summary, the use of TG2-modified COL I provides a promising new scaffold for promoting bone healing.
Asunto(s)
Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Colágeno Tipo I/química , Proteínas de Unión al GTP/química , Transglutaminasas/química , Fosfatasa Alcalina/metabolismo , Anticuerpos/farmacología , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Integrinas/inmunología , Ensayo de Materiales , Oligopéptidos/farmacología , Osteoblastos/efectos de los fármacos , Péptidos/química , Péptidos/farmacología , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transducción de Señal/efectos de los fármacosRESUMEN
Tissue transglutaminase (TG2) is a multifunctional Ca(2+)-activated protein cross-linking enzyme secreted into the extracellular matrix (ECM), where it is involved in wound healing and scarring, tissue fibrosis, celiac disease, and metastatic cancer. Extracellular TG2 can also facilitate cell adhesion important in wound healing through a nontransamidating mechanism via its association with fibronectin, heparan sulfates (HS), and integrins. Regulating the mechanism how TG2 is translocated into the ECM therefore provides a strategy for modulating these physiological and pathological functions of the enzyme. Here, through molecular modeling and mutagenesis, we have identified the HS-binding site of TG2 (202)KFLKNAGRDCSRRSSPVYVGR(222). We demonstrate the requirement of this binding site for translocation of TG2 into the ECM through a mechanism involving cell surface shedding of HS. By synthesizing a peptide NPKFLKNAGRDCSRRSS corresponding to the HS-binding site within TG2, we also demonstrate how this mimicking peptide can in isolation compensate for the RGD-induced loss of cell adhesion on fibronectin via binding to syndecan-4, leading to activation of PKCα, pFAK-397, and ERK1/2 and the subsequent formation of focal adhesions and actin cytoskeleton organization. A novel regulatory mechanism for TG2 translocation into the extracellular compartment that depends upon TG2 conformation and the binding of HS is proposed.
Asunto(s)
Matriz Extracelular/enzimología , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Transducción de Señal/fisiología , Transglutaminasas/metabolismo , Animales , Antígenos de Superficie/metabolismo , Células CHO , Dominio Catalítico/fisiología , Adhesión Celular/fisiología , Cricetinae , Proteínas de Unión al GTP , Células HEK293 , Humanos , Ratones , Modelos Moleculares , Mutagénesis/fisiología , Células 3T3 NIH , Oligopéptidos/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Sindecano-4/metabolismo , Transglutaminasas/química , Transglutaminasas/genéticaRESUMEN
Fibronectin (FN) deposition mediated by fibroblasts is an important process in matrix remodeling and wound healing. By monitoring the deposition of soluble biotinylated FN, we show that the stress-induced TG-FN matrix, a matrix complex of tissue transglutaminase (TG2) with its high affinity binding partner FN, can increase both exogenous and cellular FN deposition and also restore it when cell adhesion is interrupted via the presence of RGD-containing peptides. This mechanism does not require the transamidase activity of TG2 but is activated through an RGD-independent adhesion process requiring a heterocomplex of TG2 and FN and is mediated by a syndecan-4 and ß1 integrin co-signaling pathway. By using α5 null cells, ß1 integrin functional blocking antibody, and a α5ß1 integrin targeting peptide A5-1, we demonstrate that the α5 and ß1 integrins are essential for TG-FN to compensate RGD-induced loss of cell adhesion and FN deposition. The importance of syndecan-2 in this process was shown using targeting siRNAs, which abolished the compensation effect of TG-FN on the RGD-induced loss of cell adhesion, resulting in disruption of actin skeleton formation and FN deposition. Unlike syndecan-4, syndecan-2 does not interact directly with TG2 but acts as a downstream effector in regulating actin cytoskeleton organization through the ROCK pathway. We demonstrate that PKCα is likely to be the important link between syndecan-4 and syndecan-2 signaling and that TG2 is the functional component of the TG-FN heterocomplex in mediating cell adhesion via its direct interaction with heparan sulfate chains.
Asunto(s)
Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Proteínas de Unión al GTP/metabolismo , Integrina alfa5beta1/metabolismo , Oligopéptidos/farmacología , Transducción de Señal/fisiología , Sindecano-2/metabolismo , Sindecano-4/metabolismo , Transglutaminasas/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Células CHO , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Cricetinae , Cricetulus , Citoesqueleto/genética , Citoesqueleto/metabolismo , Matriz Extracelular/genética , Fibronectinas/genética , Proteínas de Unión al GTP/genética , Heparitina Sulfato/genética , Heparitina Sulfato/metabolismo , Humanos , Integrina alfa5beta1/genética , Ratones , Ratones Mutantes , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Sindecano-2/genética , Sindecano-4/genética , Transglutaminasas/genética , Quinasas Asociadas a rho/genética , Quinasas Asociadas a rho/metabolismoRESUMEN
The multifunctional enzyme tissue transglutaminase (TG2) is reported to both mediate and inhibit tumour progression. To elucidate these different roles of TG2, we established a series of stable-transfected mouse colon carcinoma CT26 cells expressing a catalytically active (wild type) and a transamidating-inactive TG2 (Cys277Ser) mutant. Comparison of the TG2-transfected cells with the empty vector control indicated no differences in cell proliferation, apoptosis and susceptibility to doxorubicin, which correlated with no detectable changes in the activation of the transcription factor NF-κB. TG2-transfected cells showed increased expression of integrin ß3, and were more adherent and less migratory on fibronectin than control cells. Direct interaction of TG2 with ß3 integrins was demonstrated by immunoprecipitation, suggesting that TG2 acts as a coreceptor for fibronectin with ß3 integrins. All cells expressed the same level of TGFß receptors I and II, but only cells transfected with active TG2 had increased levels of TGFß1 and matrix-deposited fibronectin, which could be inhibited by TG2 site-directed inhibitors. Moreover, only cells transfected with active TG2 were capable of inhibiting tumour growth when compared to the empty vector controls. We conclude that in this colon carcinoma model increased levels of active TG2 are unfavourable to tumour growth due to their role in activation of TGFß1 and increased matrix deposition, which in turn favours increased cell adhesion and a lowered migratory and invasive behaviour.
Asunto(s)
Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Proteínas de Unión al GTP/metabolismo , Transglutaminasas/metabolismo , Animales , Apoptosis/genética , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias del Colon/tratamiento farmacológico , Doxorrubicina/farmacología , Matriz Extracelular/metabolismo , Femenino , Fibronectinas , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/inmunología , Cadenas beta de Integrinas , Integrina beta1 , Integrina beta3 , Ratones , Ratones Endogámicos BALB C , FN-kappa B/genética , FN-kappa B/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Sindecano-4 , Factor de Crecimiento Transformador beta1/metabolismo , Transglutaminasas/genética , Transglutaminasas/inmunologíaRESUMEN
In fibrotic conditions increases in TG2 activity has been linked to an increase in the deposition of extracellular matrix proteins. Using TG2 transfected Swiss 3T3 fibroblasts expressing TG2 under the control of the tetracycline-regulated inducible promoter, we demonstrate that induction of TG2 not only stimulates an increase in collagen and fibronectin deposition but also an increase in the expression of these proteins. Increased TG2 expression in these fibroblasts led to NF-kappaB activation, resulting in the increased expression of transforming growth factor (TGF) beta(1). In addition, cells overexpressing TG2 demonstrated an increase in biologically active TGFbeta(1) in the extracellular environment. A specific site-directed inhibitor of TG abolished the NF-kappaB and TGFbeta1 activation and the subsequent elevation in the synthesis and deposition of extracellular matrix proteins, confirming that this process depends on the induction of transglutaminase activity. Treatment of TG2-induced fibroblasts with nontoxic doses of nitric oxide donor S-nitroso-N-acetylpenicillamine resulted in decreased TG2 activity and apprehension of the inactive enzyme on the cell surface. This was paralleled by a reduction in activation of NF-kappaB and TGFbeta(1) production with a subsequent decrease in collagen expression and deposition. These findings support a role for NO in the regulation of TG2 function in the extracellular environment.
Asunto(s)
Colágeno/biosíntesis , Fibrina/biosíntesis , Proteínas de Unión al GTP/biosíntesis , Óxido Nítrico/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Transglutaminasas/biosíntesis , Células 3T3 , Animales , Colágeno/genética , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibrina/genética , Proteínas de Unión al GTP/genética , Humanos , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Óxido Nítrico/genética , Donantes de Óxido Nítrico/farmacología , Proteína Glutamina Gamma Glutamiltransferasa 2 , S-Nitroso-N-Acetilpenicilamina/farmacología , Factor de Crecimiento Transformador beta1/genética , Transglutaminasas/genéticaRESUMEN
OBJECTIVE: Earlier work has demonstrated that serum autoantibodies from coeliac patients targeted against transglutaminase 2 (TG2) inhibit in vitro angiogenesis. The aim of this study was to establish whether coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology exert similar anti-angiogenic effects to serum-derived coeliac autoantibodies. In addition, we studied whether the monoclonal patient autoantibodies modulate endothelial cell TG2 activity and whether such modulation is related to the anti-angiogenic effects. MATERIAL AND METHODS: The influence of coeliac patient-derived monoclonal TG2-targeted antibodies on endothelial cell tubule formation was studied using a three-dimensional angiogenic cell culture model. Endothelial cell TG2 enzymatic activity was determined by means of a live-cell enzyme-linked immunosorbent assay. RESULTS: Coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology inhibited endothelial tubule formation and enhanced the crosslinking activity of TG2. When this enzymatic activity was inhibited using site-directed irreversible TG2 inhibitors in the presence of autoantibodies, in vitro angiogenesis reverted to the control level. CONCLUSIONS: Since we found a significant negative correlation between endothelial cell angiogenesis and TG2 activity, we suggest that the anti-angiogenic effects of coeliac patient-derived TG2-targeted autoantibodies are exerted by enhanced enzymatic activity of TG2.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Autoanticuerpos/fisiología , Enfermedad Celíaca/enzimología , Enfermedad Celíaca/inmunología , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/inmunología , Neovascularización Patológica/inmunología , Transglutaminasas/antagonistas & inhibidores , Transglutaminasas/inmunología , Análisis de Varianza , Biopsia , Western Blotting , Técnicas de Cultivo de Célula , Células Endoteliales/inmunología , Endotelio Vascular/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina A/inmunología , Proteína Glutamina Gamma Glutamiltransferasa 2RESUMEN
Celiac disease is characterized by the presence of specific autoantibodies targeted against transglutaminase 2 (TG2) in untreated patients' serum and at their production site in the small-bowel mucosa below the basement membrane and around the blood vessels. As these autoantibodies have biological activity in vitro, such as inhibition of angiogenesis, we studied if they might also modulate the endothelial barrier function. Our results show that celiac disease patient autoantibodies increase endothelial permeability for macromolecules, and enhance the binding of lymphocytes to the endothelium and their transendothelial migration when compared to control antibodies in an endothelial cell-based in vitro model. We also demonstrate that these effects are mediated by increased activities of TG2 and RhoA. Since the small bowel mucosal endothelium serves as a "gatekeeper" in inflammatory processes, the disease-specific autoantibodies targeted against TG2 could thus contribute to the pathogenic cascade of celiac disease by increasing blood vessel permeability.
Asunto(s)
Autoanticuerpos/fisiología , Enfermedad Celíaca/metabolismo , Proteínas de Unión al GTP/metabolismo , Inmunoglobulina A/fisiología , Transglutaminasas/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiopatología , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/fisiopatología , Adhesión Celular , Endotelio/metabolismo , Humanos , Linfocitos/metabolismo , Permeabilidad , Proteína Glutamina Gamma Glutamiltransferasa 2RESUMEN
Enhancement of collagen's physical characteristics has been traditionally approached using various physico-chemical methods frequently compromising cell viability. Microbial transglutaminase (mTGase), a transamidating enzyme obtained from Streptomyces mobaraensis, was used in the cross-linking of collagen-based scaffolds. The introduction of these covalent bonds has previously indicated increased proteolytic and mechanical stability and the promotion of cell colonisation. The hypothesis behind this research is that an enzymatically stabilised collagen scaffold will provide a dermal precursor with enhanced wound healing properties. Freeze-dried scaffolds, with and without the loading of a site-directed mammalian transglutaminase inhibitor to modulate matrix deposition, were applied to full thickness wounds surgically performed on rats' dorsum and explanted at three different time points (3, 7 and 21 days). Wound healing parameters such as wound closure, epithelialisation, angiogenesis, inflammatory and fibroblastic cellular infiltration and scarring were analysed and quantified using stereological methods. The introduction of this enzymatic cross-linking agent stimulated neovascularisation and epithelialisation resisting wound contraction. Hence, these characteristics make this scaffold a potential candidate to be considered as a dermal precursor.
Asunto(s)
Dermis/patología , Cicatrización de Heridas , Animales , Colágeno Tipo III/metabolismo , Dermis/metabolismo , Epitelio/irrigación sanguínea , Epitelio/metabolismo , Epitelio/patología , Fibroblastos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Dipeptide-based sulfonium peptidylmethylketones derived from 6-diazo-5-oxo-L-norleucine (DON) have been investigated as potential water-soluble inhibitors of extracellular transglutaminase. The lead compounds were prepared in four steps and exhibited potent activity against tissue transglutaminase.
Asunto(s)
Diazooxonorleucina/química , Proteínas de Unión al GTP/antagonistas & inhibidores , Transglutaminasas/antagonistas & inhibidores , Agua/química , Dominio Catalítico , Química Farmacéutica/métodos , Diseño de Fármacos , Etanol/química , Humanos , Concentración 50 Inhibidora , Cetonas/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas/métodos , Péptidos/química , Proteína Glutamina Gamma Glutamiltransferasa 2 , Solubilidad , Espectrofotometría/métodosRESUMEN
Periodontal disease is a prevalent chronic inflammatory condition characterised by an aberrant host response to a pathogenic plaque biofilm resulting in local tissue damage and frustrated healing that can result in tooth loss. Cysteine proteases (gingipains) from the key periodontal pathogen Porphyromonas gingivalis have been implicated in periodontal disease pathogenesis by inhibiting inflammation resolution and are linked with systemic chronic inflammatory conditions such as rheumatoid arthritis. Efficient clearance of apoptotic cells is essential for the resolution of inflammation and tissue restoration. Here we sought to characterise the innate immune clearance of apoptotic cells and its modulation by gingipains. We examined the capacity of gingipain-treated macrophages to migrate towards and phagocytose apoptotic cells. Lysine gingipain treatment of macrophages impaired macrophage migration towards apoptotic neutrophils. Furthermore, lysine gingipain treatment reduced surface expression levels of CD14, a key macrophage receptor for apoptotic cells, which resulted in reduced macrophage interactions with apoptotic cells. Additionally, while apoptotic cells and their derived secretome were shown to inhibit TNF-α-induced expression by P. gingivalis lipopolysaccharide, we demonstrated that gingipain preparations induced a rapid inflammatory response in macrophages that was resistant to the anti-inflammatory effects of apoptotic cells or their secretome. Taken together, these data indicate that P. gingivalis may promote the chronic inflammation seen in periodontal disease patients by multiple mechanisms, including rapid, potent gingipain-mediated inflammation, coupled with receptor cleavage leading to defective clearance of apoptotic cells and reduced anti-inflammatory responses. Thus, gingipains represent a potential therapeutic target for intervention in the management of chronic periodontal disease.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Apoptosis/fisiología , Movimiento Celular/fisiología , Cisteína Endopeptidasas/metabolismo , Macrófagos/patología , Neutrófilos/patología , Fagocitosis/fisiología , Porphyromonas gingivalis/metabolismo , Infecciones por Bacteroidaceae/metabolismo , Infecciones por Bacteroidaceae/microbiología , Infecciones por Bacteroidaceae/patología , Biopelículas/crecimiento & desarrollo , Línea Celular Tumoral , Proteasas de Cisteína/metabolismo , Cisteína-Endopeptidasas Gingipaínas , Humanos , Inflamación/metabolismo , Inflamación/microbiología , Inflamación/patología , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/metabolismo , Macrófagos/metabolismo , Neutrófilos/metabolismo , Neutrófilos/microbiología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
This study investigated the effect on the mechanical and physicochemical properties of type II collagen scaffolds after cross-linking with microbial transglutaminase (mTGase). It is intended to develop a collagen-based scaffold to be used for the treatment of degenerated intervertebral discs. By measuring the amount of epsilon-(gamma-glutamyl)lysine isodipeptide formed after cross-linking, it was determined that the optimal enzyme concentration was 0.005% (w/v). From the production of covalent bonds induced by mTGase cross-linking, the degradation resistance of type II collagen scaffolds can be enhanced. Rheological analysis revealed an almost sixfold increase in storage modulus (G') with 0.005% (w/v) mTGase cross-linked scaffolds (1.31 +/- 0.03 kPa) compared to controls (0.21 +/- 0.01 kPa). There was a significant reduction in the level of cell-mediated contraction of scaffolds with increased mTGase concentrations. Cell proliferation assays showed that mTGase crosslinked scaffolds exhibited similar cytocompatibility properties in comparison to non-cross-linked scaffolds. In summary, cross-linking type II collagen with mTGase imparted more desirable properties, making it more applicable for use as a scaffold in tissue engineering applications.
Asunto(s)
Proteínas Bacterianas/fisiología , Materiales Biocompatibles , Colágeno Tipo II/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Dipéptidos/metabolismo , Ingeniería de Tejidos , Transglutaminasas/fisiología , Células 3T3 , Animales , Proteínas Bacterianas/toxicidad , Proliferación Celular , Pollos , Colágeno Tipo II/química , Reactivos de Enlaces Cruzados/química , Dipéptidos/química , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Ratones , Streptomyces/enzimología , Transglutaminasas/toxicidadRESUMEN
Collagen, type I, is a highly abundant natural protein material which has been cross-linked by a variety of methods including chemical agents, physical heating and UV irradiation with the aim of enhancing its physical characteristics such as mechanical strength, thermal stability, resistance to proteolytic breakdown, thus increasing its overall biocompatibility. However, in view of the toxicity of residual cross-linking agents, or impracticability at large scales, it would be more useful if the collagen could be cross-linked by a milder, efficient and more practical means by using enzymes as biological catalysts. We demonstrate that on treating native collagen type I (from bovine skin) with both tissue transglutaminase (TG2; tTG) and microbial transglutaminase (mTG; Streptoverticillium mobaraense) leads to an enhancement in cell attachment, spreading and proliferation of human osteoblasts (HOB) and human foreskin dermal fibroblasts (HFDF) when compared to culture on native collagen. The transglutaminase-treated collagen substrates also showed a greater resistance to cell-mediated endogenous protease degradation than the native collagen. In addition, the HOB cells were shown to differentiate at a faster rate than on native collagen when assessed by measurement of alkaline phosphatase activity and osteopontin expression.
Asunto(s)
Colágeno/química , Reactivos de Enlaces Cruzados/farmacología , Transglutaminasas/química , Transglutaminasas/genética , Fosfatasa Alcalina/metabolismo , Animales , Materiales Biocompatibles/química , Biomarcadores/química , Catálisis , Bovinos , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Colágeno Tipo I/química , Colagenasas/química , Dipéptidos/química , Fibroblastos/metabolismo , Proteínas de Unión al GTP/química , Gelatina/química , Calor , Humanos , Osteoblastos/metabolismo , Osteopontina , Proteína Glutamina Gamma Glutamiltransferasa 2 , Sialoglicoproteínas/química , Factores de Tiempo , Rayos Ultravioleta , Cicatrización de HeridasRESUMEN
The definitive goal of this research is to develop protein-based scaffolds for use in soft tissue regeneration, particularly in the field of dermal healing. The premise of this investigation was to characterize the mechanical properties of gelatin cross-linked with microbial transglutaminase (mTGase) and to investigate the cytocompatibility of mTGase cross-linked gelatin. Dynamic rheological analysis revealed a significant increase in the storage modulus and thermal stability of gelatin after cross-linking with mTGase. Static, unconfined compression tests showed an increase in Young's modulus of gelatin gels after mTGase cross-linking. A comparable increase in gel strength was observed with 0.03% mTGase and 0.25% glutaraldehyde cross-linked gelatin gels. In vitro studies using 3T3 fibroblasts indicated cytotoxicity at a concentration of 0.05% mTGase after 72 h. However, no significant inhibition of cell proliferation was seen with cells grown on lower concentrations of mTGase cross-linked gelatin substrates. The mechanical improvement and cytocompatibility of mTGase cross-linked gelatin suggests mTGase has potential for use in stabilizing gelatin gels for tissue-engineering applications.
Asunto(s)
Gelatina/química , Gelatina/metabolismo , Transglutaminasas/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Gelatina/farmacología , Glutaral/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Ratones , Células 3T3 NIH , Porcinos , Temperatura , Factores de Tiempo , Ingeniería de Tejidos/métodosRESUMEN
New peptidic water-soluble inhibitors are reported. In addition to the carboxylate moiety, a new polar warhead was explored. Depending on the size of its substituents, the newly appended imidazolium scaffold designed to enhance the hydrophilic character of the inhibitors could induce a good inhibition for tissue transglutaminase (TG2) and blood coagulation factor XIIIa (FXIIIa). Correlated with the narrow tunnel that hosts the target catalytic cysteine residue, the various modulations suggest a bent conformation of the ligands as the binding pattern mode. Analogues in the dialkylsulfonium series were also tested and showed specificity for TG2 over FXIIIa.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Imidazoles/química , Péptidos/química , Péptidos/farmacología , Transglutaminasas/antagonistas & inhibidores , Agua/química , Sitios de Unión , Proteínas de Unión al GTP , Humanos , Modelos Moleculares , Conformación Proteica , Proteína Glutamina Gamma Glutamiltransferasa 2 , Solubilidad , Transglutaminasas/químicaRESUMEN
Central venous catheters (CVCs) are being utilized with increasing frequency in intensive care and general medical wards. In spite of the extensive experience gained in their application, CVCs are related to the long-term risks of catheter sheath formation, infection, and thrombosis (of the catheter or vessel itself) during catheterization. Such CVC-related-complications are associated with increased morbidity, mortality, duration of hospitalization, and medical care cost [1]. The present study incorporates a novel group of Factor XIIIa (FXIIIa, plasma transglutaminase) inhibitors into a lubricious silicone elastomer in order to generate an optimized drug delivery system whereby a secondary sustained drug release profile occurs following an initial burst release for catheters and other medical devices. We propose that the incorporation of FXIIIa inhibitors into catheters, stents, and other medical implant devices would reduce the incidence of catheter sheath formation, thrombotic occlusion, and associated staphylococcal infection. This technique could be used as a local delivery system for extended release with an immediate onset of action for other poorly aqueous soluble compounds.
Asunto(s)
Infecciones Relacionadas con Catéteres/prevención & control , Dipéptidos/farmacología , Sistemas de Liberación de Medicamentos , Factor XIIIa/antagonistas & inhibidores , Sulfonamidas/farmacología , Catéteres de Permanencia/efectos adversos , Catéteres Venosos Centrales/efectos adversos , Dipéptidos/administración & dosificación , Equipos y Suministros/efectos adversos , Humanos , Elastómeros de Silicona/química , Sulfonamidas/administración & dosificaciónRESUMEN
Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides. In our previous work, we have shown the tethering of laminin-332 α3 chain to type I collagen scaffold using microbial transglutaminase (mTGase), promotes cell adhesion, migration, and proliferation. In this study, we evaluated the wound healing properties of tailored laminin-332 α3 chain (peptide A: PPFLMLLKGSTR) tethered to a type I collagen scaffold using mTGase by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide B: PPFLMLLKGSTREAQQIVM) or lysine (peptide C: PPFLMLLKGSTRKKKKG) in rat full-thickness wound model at two different time points (7 and 21 days). Histological evaluations were assessed for wound closure, epithelialization, angiogenesis, inflammatory, fibroblastic cellular infiltrations, and quantified using stereological methods (p < 0.05). Peptide A and B tethered to collagen scaffold using mTGase stimulated neovascularization, decreased the inflammatory cell infiltration and prominently enhanced the fibroblast proliferation which significantly accelerated the wound healing process. We conclude that surface modification by incorporating motif of laminin-332 α3 chain (peptide A: PPFLMLLK GSTR) domain and transglutaminase substrate to the laminin-332 α3 chain (peptide B: PPFLMLLKGSTREAQQIVM) using mTGase may be a potential candidate for tissue engineering applications and skin regeneration.
Asunto(s)
Moléculas de Adhesión Celular/farmacología , Andamios del Tejido/química , Cicatrización de Heridas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Bovinos , Moléculas de Adhesión Celular/química , Tamaño de la Célula/efectos de los fármacos , Colágenos Fibrilares/química , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Inflamación/patología , Masculino , Datos de Secuencia Molecular , Neovascularización Fisiológica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transglutaminasas/metabolismo , KalininaRESUMEN
Liver fibrosis and its end-stage disease cirrhosis are a main cause of mortality and morbidity worldwide. Thus far, there is no efficient pharmaceutical intervention for the treatment of liver fibrosis. Liver fibrosis is characterized by excessive accumulation of the extracellular matrix (ECM) proteins. Transglutaminase (TG)-mediated covalent cross-linking has been implicated in the stabilization and accumulation of ECM in a number of fibrotic diseases. Thus, the use of tissue TG2 inhibitors has potential in the treatment of liver fibrosis. Recently, we introduced a novel group of site-directed irreversible specific inhibitors of TGs. Here, we describe the development of a liposome-based drug-delivery system for the site-specific delivery of these TG inhibitors into the liver. By using anionic or neutral-based DSPC liposomes, the TG inhibitor can be successfully incorporated into these liposomes and delivered specifically to the liver. Liposomes can therefore be used as a potential carrier system for site-specific delivery of the TG2 inhibitors into the liver, opening up a potential new avenue for the treatment of liver fibrosis and its end-stage disease cirrhosis.
Asunto(s)
Portadores de Fármacos/química , Inhibidores Enzimáticos/administración & dosificación , Proteínas de Unión al GTP/antagonistas & inhibidores , Cirrosis Hepática/tratamiento farmacológico , Transglutaminasas/antagonistas & inhibidores , Animales , Cromatografía en Gel , Composición de Medicamentos , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Liposomas , Cirrosis Hepática/enzimología , Masculino , Ratones , Terapia Molecular Dirigida , Especificidad de Órganos , Tamaño de la Partícula , Fosfatidilcolinas/química , Fosfatidilserinas/química , Proteína Glutamina Gamma Glutamiltransferasa 2 , Propiedades de Superficie , Distribución TisularRESUMEN
AIMS To demonstrate the potential use of in vitro poly(lactic-co-glycolic acid) (PLGA) microparticles in comparison with triamcinolone suspension to aid visualisation of vitreous during anterior and posterior vitrectomy. METHODS PLGA microparticles (diameter 10-60 microm) were fabricated using single and/or double emulsion technique(s) and used untreated or following the surface adsorption of a protein (transglutaminase). Particle size, shape, morphology and surface topography were assessed using scanning electron microscopy (SEM) and compared with a standard triamcinolone suspension. The efficacy of these microparticles to enhance visualisation of vitreous against the triamcinolone suspension was assessed using an in vitro set-up exploiting porcine vitreous. RESULTS Unmodified PLGA microparticles failed to adequately adhere to porcine vitreous and were readily washed out by irrigation. In contrast, modified transglutaminase-coated PLGA microparticles demonstrated a significant improvement in adhesiveness and were comparable to a triamcinolone suspension in their ability to enhance the visualisation of vitreous. This adhesive behaviour also demonstrated selectivity by not binding to the corneal endothelium. CONCLUSION The use of transglutaminase-modified biodegradable PLGA microparticles represents a novel method of visualising vitreous and aiding vitrectomy. This method may provide a distinct alternative for the visualisation of vitreous whilst eliminating the pharmacological effects of triamcinolone acetonide suspension.
Asunto(s)
Glicolatos/química , Microesferas , Cuerpo Vítreo/patología , Adhesividad , Humanos , Técnicas In Vitro , Ácido Láctico , Microscopía Electrónica de Rastreo , Tamaño de la Partícula , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Triamcinolona , Vitrectomía/métodosRESUMEN
Cell adhesion peptide regulates various cellular functions like proliferation, attachment, and spreading. The cellular response to laminin peptide (PPFLMLLKGSTR), a motif of laminin-5 alpha3 chain, tethered to type I collagen, crosslinked using microbial transglutaminase (mTGase) was investigated. mTGase is an enzyme that initiates crosslinking by reacting with the glutamine and lysine residues on the collagen fibers stabilizing the molecular structure. In this study that tethering of the laminin peptide in a mTGase crosslinked collagen scaffold enhanced cell proliferation and attachment. Laminin peptide tethered crosslinked scaffold showed unaltered cell morphology of 3T3 fibroblasts when compared with collagen and crosslinked scaffold. The triple helical structure of collagen remained unaltered by the addition of laminin peptide. In addition a dose-dependent affinity of the laminin peptide towards collagen was seen. The degree of crosslinking was measured by amino acid analysis, differential scanning calorimeter and fourier transform infrared spectroscopy. Increased crosslinking was observed in mTGase crosslinked group. mTGase crosslinking showed higher shrinkage temperature. There was alteration in the fibrillar architecture due to the crosslinking activity of mTGase. Hence, the use of enzyme-mediated linking shows promise in tethering cell adhesive peptides through biodegradable scaffolds.