Transglutaminase inhibition ameliorates experimental diabetic nephropathy

Diabetic nephropathy is characterized by excessive extracellular matrix accumulation resulting in renal scarring and end-stage renal disease. Previous studies have suggested that transglutaminase type 2, by formation of its protein crosslink product epsilon-(gamma-glutamyl)lysine, alters extracellular matrix homeostasis, causing basement membrane thickening and expansion of the mesangium and interstitium. To determine whether transglutaminase inhibition can slow the progression of chronic experimental diabetic nephropathy over an extended treatment period, the inhibitor NTU281 was given to uninephrectomized streptozotocin-induced diabetic rats for up to 8 months. Effective transglutaminase inhibition significantly reversed the increased serum creatinine and albuminuria in the diabetic rats. These improvements were accompanied by a fivefold decrease in glomerulosclerosis and a sixfold reduction in tubulointerstitial scarring. This was associated with reductions in collagen IV accumulation by 4 months, along with reductions in collagens I and III by 8 months. This inhibition also decreased the number of myofibroblasts, suggesting that tissue transglutaminase may play a role in myofibroblast transformation. Our study suggests that transglutaminase inhibition ameliorates the progression of experimental diabetic nephropathy and can be considered for clinical application.

Tissue transglutaminase (TG2) - a wound response enzyme

Repair of tissue after injury depends on a series of concerted but overlapping events including, inflammation, re-epithelialization, neovascularization and synthesis and stabilization of a fibrous extracellular matrix (ECM) that is remodeled to emulate normal tissue over time. Particular members of the transglutaminase (TG) family are upregulated during wound healing and act as a novel class of wound-healing mediators during the repair process. This group of enzymes which crosslink proteins via epsilon(gamma-glutamyl) lysine bridges are involved in wound healing through their ability to stabilize proteins and also by regulating the behavior of a wide variety of cell types that are recruited to the damaged area in order to carry out tissue repair. In this article we discuss the function of the most widely expressed member of the TG family "tissue transglutaminase" (TG2) in wound repair. Using both early and recent evidence from the literature we demonstrate how the multifunctional TG2 affects the stability of the ECM, cell-ECM interactions and as a consequence cell behavior within the different phases of wound healing, and highlight how TG2 itself might be exploited for therapeutic use.

Importance of tissue transglutaminase in repair of extracellular matrices and cell death of dermal fibroblasts after exposure to a solarium ultraviolet A source

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.

Tailored laminin-332 alpha3 sequence is tethered through an enzymatic linker to a collagen scaffold to promote cellular adhesion

Surface modification techniques have been used to develop biomimetic scaffolds by incorporating cell adhesion peptides, which facilitates cell adhesion, migration and proliferation. In this study, we evaluated the cell adhesion properties of a tailored laminin-332 alpha3 chain tethered to a type I collagen scaffold using microbial transglutaminase (mTGase) by incorporating transglutaminase substrate peptide sequences containing either glutamine (peptide A: PPFLMLLKGSTREAQQIVM) or lysine (peptide B: PPFLMLLKGSTRKKKKG). The degree of cross-linking was studied by amino acid analysis following proteolytic digestion and the structural changes in the modified scaffold further investigated using Fourier transform infrared spectroscopy and atomic force microscopy. Fibroblasts were used to evaluate the cellular behaviour of the functionalized collagen scaffold. mTGase supports cell growth but tethering of peptide A and peptide B to the mTGase cross-linked collagen scaffold caused a significant increase in cell proliferation when compared with native and mTGase cross-linked collagen scaffolds. Both peptides enabled cell-spreading, attachment and normal actin cytoskeleton organization with slight increase in the cell proliferation was observed in peptide A when compared with the peptide B and mTGase cross-linked scaffold. An increase in the amount of epsilon(gamma-glutamyl) lysine isopeptide was observed in peptide A conjugated scaffolds when compared with peptide B conjugated scaffolds, mTGase cross-linked scaffold without peptide. Changes in D-spacing were observed in the cross-linked scaffolds with tethered peptides. These results demonstrate that mTGase can play a bifunctional role in both conjugation of the glutamine and lysine containing peptide sequences and also in the cross-linking of the collagen scaffold, thus providing a suitable substrate for cell growth.

Inhibition of transglutaminase activity reduces extracellular matrix accumulation induced by high glucose levels in proximal tubular epithelial cells

Diabetic nephropathy affects 30-40% of diabetics leading to end-stage kidney failure through progressive scarring and fibrosis. Previous evidence suggests that tissue transglutaminase (tTg) and its protein cross-link product epsilon(gamma-glutamyl)lysine contribute to the expanding renal tubulointerstitial and glomerular basement membranes in this disease. Using an in vitro cell culture model of renal proximal tubular epithelial cells we determined the link between elevated glucose levels with changes in expression and activity of tTg and then, by using a highly specific site directed inhibitor of tTg (1,3-dimethyl-2[(oxopropyl)thio]imidazolium), determined the contribution of tTg to glucose-induced matrix accumulation. Exposure of cells to 36 mm glucose over 96 h caused an mRNA-dependent increase in tTg activity with a 25% increase in extracellular matrix (ECM)-associated tTg and a 150% increase in ECM epsilon(gamma-glutamyl)lysine cross-linking. This was paralleled by an elevation in total deposited ECM resulting from higher levels of deposited collagen and fibronectin. These were associated with raised mRNA for collagens III, IV, and fibronectin. The specific site-directed inhibitor of tTg normalized both tTg activity and ECM-associated epsilon(gamma-glutamyl)lysine. Levels of ECM per cell returned to near control levels with non-transcriptional reductions in deposited collagen and fibronectin. No changes in transforming growth factor beta1 (expression or biological activity) occurred that could account for our observations, whereas incubation of tTg with collagen III indicated that cross-linking could directly increase the rate of collagen fibril/gel formation. We conclude that Tg inhibition reduces glucose-induced deposition of ECM proteins independently of changes in ECM and transforming growth factor beta1 synthesis thus opening up its possible application in the treatment other fibrotic and scarring diseases where tTg has been implicated.

Sequence analysis and distribution in Salmonella enterica serovars of IS3-like elements

The genome of Salmonella enterica serovar Enteritidis was shown to possess three IS3-like insertion elements, designated IS1230A, B and C, and each was cloned and their respective deoxynucleotide sequences determined. Mutations in elements IS1230A and B resulted in frameshifts in the open reading frames that encoded a putative transposase to be inactive. IS1230C was truncated at nucleotide 774 relative to IS1230B and therefore did not possess the 3' terminal inverted repeat. The three IS1230 derivatives were closely related to each other based on nucleotide sequence similarity. IS1230A was located adjacent to the sef operon encoding SEF14 fimbriae located at minute 97 of the genome of S. Enteritidis. IS1230B was located adjacent to the umuDC operon at minute 42.5 on the genome, itself located near to one terminus of an 815-kb genome inversion of S. Enteritidis relative to S. Typhimurium. IS1230C was located next to attB, the bacteriophage P22 attachment site, and proB, encoding gamma-glutamyl phosphate reductase. A truncated 3' remnant of IS1230, designated IS1230T, was identified in a clinical isolate of S. Typhimurium DT193 strain 2391. This element was located next to attB adjacent to which were bacteriophage P22-like sequences. Southern hybridisation of total genomic DNA from eighteen phage types of S. Enteritidis and eighteen definitive types of S. Typhimurium showed similar, if not identical, restriction fragment profiles in the respective serovars when probed with IS1230A.

Transglutaminases as tanning agents for the leather industry

This study was aimed at determining whether the protein crosslinking enzymes, transglutaminases, had the potential to be used as tanning agents, using native bovine hide and purified soluble rat tail collagen as real and model substrates, respectively. We demonstrate that transglutaminases (TGs) were capable of covalently crosslinking collagen molecules together such that on average every collagen molecule contained at least one epsilon(gamma-glutamyl)lysine crosslink. However, transglutaminase-mediated crosslinking did not affect the denaturation temperature of either native bovine hide or soluble rat tail collagens when used in isolation or together with other proteins and bifunctional diamines as crosslinking facilitators. In an initial study into the effect of TG-mediated crosslinking on the tensile strength of chrome-tanned bovine hide, such crosslinking led to a 30 per cent decrease in tensile strength. Despite a change in the gel melting point mediated by epsilon(gamma-glutamyl)lysine crosslinking, the use of transglutaminases as alternative tanning agents seems unlikely given the present data.

Tissue transglutaminase:a mediator and predictor of chronic allograft nephropathy?

Background. The precise mechanisms underlying the development of chronic allograft nephropathy (CAN) and the associated renal fibrosis remain uncertain. The protein-crosslinking enzyme, tissue transglutaminase (tTg), has recently been implicated in renal fibrosis. Methods. We investigated the involvement of tTg and its crosslink product, [epsilon]-([gamma]-glutamyl) lysine, in 23 human kidney allografts during the early posttransplantation period and related these to changes of CAN that developed in 8 of them. Sequential biopsies were investigated using immunohistochemical, immunofluorescence, and in situ enzyme activity techniques. Results. From implantation, tTg (+266%) and [epsilon]-([gamma]-glutamyl) lysine crosslink (+256.3%) staining increased significantly (P <0.001) in a first renal biopsy performed within 3 months from transplantation. This was paralleled by elevated tTg in situ activity. The eight patients who developed CAN had further increases in immunostainable tTg (+197.2%, P <0.001) and [epsilon]-([gamma]-glutamyl) lysine bonds (+465%, P <0.01) that correlated with interstitial fibrosis (r=0.843, P =0.009 and r=0.622, P =0.05, respectively). The staining for both was predominantly located within the mesangium and the renal interstitium. Both implantation and first biopsies showed tTg and [epsilon]-([gamma]-glutamyl) lysine crosslinking levels in patients who developed CAN to be twice the levels of those with stable renal function. Cox regression analysis suggested the intensity of the early tTg staining was a better predictor of inferior allograft survival that other histologic markers (hazard ratio=4.48, P =0.04). Conclusions. tTg and [epsilon]-([gamma]-glutamyl) lysine crosslink correlated with the initiation and progression of scarring on sequential biopsies from renal-allograft recipients who experienced CAN. Elevated tTg may offer an early predictor of the development of CAN, whereas tTg manipulation may be an attractive therapeutic target

Transglutaminases:nature's biological glues

Transglutaminases (Tgases) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of isopeptide bonds. This occurs either through protein cross-linking via epsilon-(gamma-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation, and their accumulation is found in a number of tissues and processes where such properties are important, including skin, hair, blood clotting and wound healing. However, deregulation of enzyme activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin. In the present review we detail the structural and regulatory features important in mammalian Tgases, with particular focus on the ubiquitous type 2 tissue enzyme. Physiological roles and substrates are discussed with a view to increasing and understanding the pathogenesis of the diseases associated with transglutaminases. Moreover the ability of these enzymes to modify proteins and act as biological glues has not gone unnoticed by the commercial sector. As a consequence, we have included some of the present and future biotechnological applications of this increasingly important group of enzymes.