Russell Collighan

Transglutaminase 2 cross-linking of matrix proteins: biological significance and medical applications

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.

Synthesis of potent water-soluble tissue transglutaminase inhibitors

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.

RGD-independent Cell Adhesion via a Tissue Transglutaminase-Fibronectin Matrix Promotes Fibronectin Fibril Deposition and Requires Syndecan-4/2 and α5β1 Integrin Co-signaling

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 β1 integrin co-signaling pathway. By using α5 null cells, β1 integrin functional blocking antibody, and a α5β1 integrin targeting peptide A5-1, we demonstrate that the α5 and β1 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.

Increased TG2 Expression Can Result in Induction of Transforming Growth Factor β1, Causing Increased Synthesis and Deposition of Matrix Proteins, Which Can Be Regulated by Nitric Oxide

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-κB activation, resulting in the increased expression of transforming growth factor (TGF) β1. In addition, cells overexpressing TG2 demonstrated an increase in biologically active TGFβ1 in the extracellular environment. A specific site-directed inhibitor of TG abolished the NF-κB and TGFβ1 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-κB and TGFβ1 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.

In vitro characterization of a collagen scaffold enzymatically cross-linked with a tailored elastin-like polymer

Collagen, the main structural component of the extracellular matrix (ECM), provides tensile stiffness to different structures and organs against rupture. However, collagen tissue-engineered implants are hereto still lacking in mechanical strength. Attempts to create stiffer scaffolds have resulted in increased brittleness of the material, reducing the versatility of the original component. The hypothesis behind this research is that the introduction of an elastic element in the scaffold will enhance the mechanical properties of the collagen-based scaffolds, as elastin does in the ECM to prevent irreversible deformation. In this study, an elastin-like polymer (ELP) designed and synthesized using recombinant DNA methodology is used with the view to providing increased proteolytic resistance and increased functionality to the scaffolds by carrying specific sequences for microbial transglutaminase cross-linking, endothelial cell adhesion, and drug delivery. Evaluation of the effects that cross-linking ELP-collagen has on the physicochemical properties of the scaffold such as porosity, presence of cross-linking, thermal behavior, and mechanical strength demonstrated that the introduction of enzymatically resistant covalent bonds between collagen and ELP increases the mechanical strength of the scaffolds in a dose-dependent manner without significantly affecting the porosity or thermal properties of the original scaffold. Importantly, the scaffolds also showed selective behavior, in a dose (ELP)-dependent manner toward human umbilical vein endothelial cells and smooth muscle cells when compared to fibroblasts.

Celiac disease IgA modulates vascular permeability in vitro through the activity of transglutaminase 2 and RhoA

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.

Characterization of Heparin-binding Site of Tissue Transglutaminase ITS IMPORTANCE IN CELL SURFACE TARGETING, MATRIX DEPOSITION, AND CELL SIGNALING

Background: TG2 is a multifunctional matrix protein and cross-linking enzyme. Results: Identification and mutation of its heparan sulfate (HS)-binding site blocks matrix deposition of TG2 as do inhibitors of syndecan shedding. Conclusion: Coordinated binding to cell surface heparan sulfates facilitates TG2 cell surface trafficking and deposition into the ECM. Significance: Blocking heparan sulfate binding provides an avenue for regulating the pathological roles of the enzyme. Tissue transglutaminase (TG2) is a multifunctional Ca2+-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 202KFLKNAGRDCSRRSSPVYVGR222. 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.

Epithelial transport and deamidation of gliadin peptides: a role for coeliac disease patient immunoglobulin A.

In coeliac disease, the intake of dietary gluten induces small‐bowel mucosal damage and the production of immunoglobulin (Ig)A class autoantibodies against transglutaminase 2 (TG2). We examined the effect of coeliac patient IgA on the apical‐to‐basal passage of gluten‐derived gliadin peptides p31–43 and p57–68 in intestinal epithelial cells. We demonstrate that coeliac IgA enhances the passage of gliadin peptides, which could be abolished by inhibition of TG2 enzymatic activity. Moreover, we also found that both the apical and the basal cell culture media containing the immunogenic gliadin peptides were able to induce the proliferation of deamidation‐dependent coeliac patient‐derived T cells even in the absence of exogenous TG2. Our results suggest that coeliac patient IgA could play a role in the transepithelial passage of gliadin peptides, a process during which they might be deamidated.

Recent advances in the development of tissue transglutaminase (TG2) inhibitors

Tissue transglutaminase (TG2) is a Ca2+-dependent enzyme and probably the most ubiquitously expressed member of the mammalian transglutaminase family. TG2 plays a number of important roles in a variety of biological processes. Via its transamidating function, it is responsible for the cross-linking of proteins by forming isopeptide bonds between glutamine and lysine residues. Intracellularly, Ca2+ activation of the enzyme is normally tightly regulated by the binding of GTP. However, upregulated levels of TG2 are associated with many disease states like celiac sprue, certain types of cancer, fibrosis, cystic fibrosis, multiple sclerosis, Alzheimer’s, Huntington’s and Parkinson’s disease. Selective inhibitors for TG2 both cell penetrating and non-cell penetrating would therefore serve as novel therapeutic tools for the treatment of these disease states. Moreover, they would provide useful tools to fully elucidate the cellular mechanisms TG2 is involved in and help comprehend how the enzyme is regulated at the cellular level. The current paper is intended to give an update on the recently discovered classes of TG2 inhibitors along with their structure–activity relationships. The biological properties of these derivatives, in terms of both activity and selectivity, will also be reported in order to translate their potential for future therapeutic developments.

Inhibition of transglutaminase 2 enzymatic activity ameliorates the anti-angiogenic effects of coeliac disease autoantibodies

Abstract 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.