Protein Cross-linking Activity Research Articles

Evidences for a role of protein cross-links in transglutaminase-related disease

Transglutaminases (TGs) are a large family of related and ubiquitous enzymes that catalyze the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. Considerable and intense progress has been made in the understanding of the chemistry, molecular biology and cell biology of TGs. The knowledge that very different physiological and pathological processes are dependent on the presence of adequate levels of these cross-linking enzymes and on the amount of both free and protein-conjugated polyamines by TG, has generated an incredible amount of original research and review articles. It is clear that TG-mediated reactions are essential for some biological processes, such as blood coagulation, skin barrier formation and extracellular matrix assembly, but may also be involved in pathogenetic mechanisms responsible for several human diseases, such as cancer, AIDS, neurodegenerative disorders, celiac disease, and eye lens opacification. We present here a comprehensive review of recent insights into the pathophysiology of TGs related to their protein cross-linking activity.

Opposing Effects of Two Tissue Transglutaminase Protein Isoforms in Neuroblastoma Cell Differentiation

We have demonstrated previously that the Myc oncoprotein blocks cancer cell differentiation by forming a novel transcriptional repressor complex with histone deacetylase and inhibiting gene transcription of tissue transglutaminase (TG2). Moreover, induction of TG2 gene transcription and transamidase activity is essential for the differentiating effects of retinoids in cancer cells. Here, we show that two structurally distinct TG2 protein isoforms, the full-length (TG2-L) and the short form (TG2-S), exert opposing effects on cell differentiation. Repression of TG2-L with small interfering RNA, which did not affect TG2-S expression, induced dramatic neuritic differentiation in neuroblastoma cells. In contrast, overexpression of TG2-S or a GTP-binding-deficient mutant of TG2-L (R580A), both of which lack the GTP-binding Arg-580 residue, induced neuroblastoma cell differentiation, which was blocked by an inhibitor of transamidase activity. Whereas N-Myc repressed and retinoid activated both TG2 isoforms, repression of TG2-L, but not simultaneous repression of TG2-L and TG2-S, enhanced neuroblastoma cell differentiation due to N-Myc small interfering RNA or retinoid. Moreover, suppression of vasoactive intestinal peptide (VIP) expression alone induced neuroblastoma cell differentiation, and VIP was up-regulated by TG2-L, but not TG2-S. Taken together, our data indicate that TG2-L and TG2-S exert opposite effects on cell differentiation due to differences in GTP binding and modulation of VIP gene transcription. Our findings highlight the potential importance of repressing the GTP binding activity of TG2-L or activating the transamidase activity of TG2-L or TG2-S for the treatment of neuroblastoma, and possibly also other Myc-induced malignancies, and for enhancing retinoid anticancer effects.

Regulation of ATPase activity of transglutaminase 2 by MT1‐MMP: Implications for mineralization of MC3T3‐E1 osteoblast cultures

A pro-mineralization function for transglutaminase 2 (TG2) has been suggested in numerous studies related to bone, cartilage, and vascular calcification. TG2 is an enzyme which can perform protein crosslinking functions, or act as a GTPase/ATPase depending upon different stimuli. We have previously demonstrated that TG2 can act as an ATPase in a Ca(2+)-rich environment and that it can regulate phosphate levels in osteoblast cultures. In this study, we investigate the role MT1-MMP in regulating the ATPase activity of TG2. We report that proteolytic cleavage of TG2 by MT1-MMP in vitro results in nearly a 3-fold increase in the ATPase activity of TG2 with a concomitant reduction in its protein-crosslinking activity. We show that MC3T3-E1 osteoblasts secreted full-length TG2 and major smaller fragments of 66 and 56 kDa, the latter having ATP-binding abilities. MT1-MMP inhibition by a neutralizing antibody suppressed mineralization of osteoblast cultures to 35% of control, and significantly reduced phosphate levels in conditioned medium (CM). Furthermore, MT1-MMP inhibition abolished two of TG2 fragments in the cultures, one of which, the 56-kDa fragment, has ATPase activity. Neutralization of MT1-MMP at early phases of mineralization significantly reduced mineral deposition, but had no effect in later phases implying MT1-MMP and TG2 might contribute to the initiation of mineralization. The cleavage of TG2 by MT1-MMP likely occurs on the cell surface/pericellular matrix where MT1-MMP and TG2 were co-localized. Based on these data, we propose that MT1-MMP modulates the extracellular function TG2 as part of a regulatory mechanism activates the pro-mineralization function of TG2.

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.

Characterization of a Novel Interaction between ELMO1 and ERM Proteins

ERMs are closely related proteins involved in cell migration, cell adhesion, maintenance of cell shape, and formation of microvilli through their ability to cross-link the plasma membrane with the actin cytoskeleton. ELMO proteins are also known to regulate actin cytoskeleton reorganization through activation of the small GTPbinding protein Rac via the ELMO-Dock180 complex. Here we showed that ERM proteins associate directly with ELMO1 as purified recombinant proteins in vitro and at endogenous levels in intact cells. We mapped ERM binding on ELMO1 to the N-terminal 280 amino acids, which overlaps with the region required for binding to the GTPase RhoG, but is distinct from the C-terminal Dock180 binding region. Consistent with this, ELMO1 could simultaneously bind both radixin and Dock180, although radixin did not alter Rac activation via the Dock180-ELMO complex. Most interestingly, radixin binding did not affect ELMO binding to active RhoG and a trimeric complex of active RhoG-ELMO-radixin could be detected. Moreover, the three proteins colocalized at the plasma membrane. Finally, in contrast to most other ERM-binding proteins, ELMO1 binding occurred independently of the state of radixin C-terminal phosphorylation, suggesting an ELMO1 interaction with both the active and inactive forms of ERM proteins and implying a possible role of ELMO in localizing or retaining ERM proteins in certain cellular sites. Together these data suggest that ELMO1-mediated cytoskeletal changes may be coordinated with ERM protein crosslinking activity during dynamic cellular functions.

Transglutaminases in mineralized tissues

Bone development and formation during embryogenesis as well as postnatally during bone remodeling is a complex process controlled systemically and locally by hormones, growth factors and matrix molecules. Transglutaminases (TGases) are the protein cross-linking enzymes, which have long been implicated in bone development and formation. Two members of TGase family, TG2 (also called tissue transglutaminase) and FXIIIA (the enzymatic A subunit of coagulation factor XIII), are expressed in chondrocytes and osteoblasts. The results of analyses in vivo and in vitro accumulated to date indicate an important role of these enzymes in promoting chondrocyte and osteoblast differentiation and matrix mineralization. These effects could be mediated by protein cross-linking activity of TGases, by GTPase activity of TG2 or via non-catalytic signaling effects. The aim of this review is to summarize the available data regarding the expression, localization and activity of TG2 and FXIIIA in mineralizing tissues and to discuss a number of mechanisms by which TGases could exert their promineralizing effects.

Identification of a Novel Recognition Sequence for Fibronectin within the NH2-terminal β-Sandwich Domain of Tissue Transglutaminase

Tissue transglutaminase belongs to the multigene transglutaminase family of Ca2+-dependent protein cross-linking enzymes. Unlike other transglutaminases, it is involved in cell-matrix interactions and serves as an adhesion co-receptor for fibronectin. Previous work established that the fibronectin-binding motif(s) is located within the NH2-terminal proteolytic fragment of the protein consisting of residues 1-272. Here we identify a novel fibronectin recognition site within this sequence of tissue transglutaminase. Substitution of individual domains of tissue transglutaminase with those from homologous factor XIIIA showed that the major fibronectin-binding site is present within the first beta-sandwich domain of the protein. Experiments with deletion mutants of the first domain revealed that amino acids 81-140 of tissue transglutaminase are involved in fibronectin binding. Using synthetic peptides encompassing this region, we found that the peptide 88WTATVVDQQDCTLSLQLTT106 inhibited the interaction of tissue transglutaminase with fibronectin and decreased transglutaminase-dependent cell adhesion and spreading. In the three-dimensional structure of the first domain, amino acids 88-106 comprise an extended hairpin formed by antiparallel beta strands 5 and 6. Mutations of Asp94 and Asp97 within the beta5/beta6 hairpin to Ala significantly reduced the affinity of tissue transglutaminase for fibronectin, indicating that these residues are critical for fibronectin binding. Identification of the fibronectin-binding site on tissue transglutaminase will help to dissect the role of this protein in cell-matrix interactions.

Transglutaminase 2 in the balance of cell death and survival

Transglutaminase 2 (TG2), a multifunctional enzyme with Ca 2+-dependent protein crosslinking activity and GTP-dependent G protein functions, is often upregulated in cells undergoing apoptosis. In cultured cells TG2 may exert both pro- and anti-apoptotic effects depending upon the type of cell, the kind of death stimuli, the intracellular localization of the enzyme and the type of its activities switched on. The majority of data support the notion that transamidation by TG2 can both facilitate and inhibit apoptosis, while the GTP-bound form of the enzyme generally protects cells against death. In vivo studies confirm the Janus face of TG2 in the initiation of the apoptotic program. In addition, they reveal a further role: the prevention of inflammation, tissue injury and autoimmunity once the apoptosis has already been initiated. This function of TG2 is partially achieved by being expressed and activated also in macrophages digesting apoptotic cells and mediating a crosstalk between dying and phagocytic cells.

Nucleotide binding by the erythrocyte transglutaminase/Gh protein, probed with fluorescent analogs of GTP and GDP.

GTP is known to be a potent inhibitor of the protein crosslinking activity of transglutaminase (TG), probably the most abundant G protein in the human red cell. Nucleotide binding to TG was examined by fluorescence spectroscopy and anisotropy in mixtures of TG with methylanthraniloyl analogs of GTP and GDP. A characteristic feature was the appearance of a major energy transfer band (lambda(exc, max) = 290 nm, lambda(em) = 444 nm) from protein tryptophans to the bound nucleotides. Quenching of the bound fluorophore (lambda(exc) = 360 nm, lambda(em) = 444 nm) by acrylamide was barely different from that of free ligand. However, major changes were observed in anisotropy, which was used to demonstrate a facile exchange between bound and free nucleotides and to evaluate affinity constants for the binding of methylanthraniloyl GTP and GDP to TG.

Analysis of DNA–protein crosslinking activity of malondialdehyde in vitro

In an attempt to identify endogenous chemicals producing DNA–protein crosslinks, we have studied in vitro crosslinking potential of malondialdehyde, a bifunctional chemical that is ubiquitously formed as a product of lipid peroxidation of polyunsaturated fatty acids. We have found that malondialdehyde readily forms crosslinks between DNA and histones under physiological ionic and pH conditions. Formation of DNA–protein crosslinks was limited to proteins that were able to bind to DNA. Malondialdehyde failed to form DNA–protein crosslinks when histone binding to DNA was prevented by elevated ionic strength or when bovine serum albumin was used in the reaction mixture. Malondialdehyde-produced DNA–histone crosslinks were relatively stable at 37°C with t 1/2=13.4 days. Crosslinking of histones to DNA proceeds through the initial formation of protein adduct followed by reaction with DNA. Modification of DNA by malondialdehyde does not lead to a subsequent crosslinking of proteins. Significant formation of DNA–protein crosslinks was also registered in isolated kidney and liver nuclei treated with malondialdehyde. Based on its reactivity and stability of the resulting crosslinks, it is suggested that malondialdehyde could be one of the significant sources of endogenous DNA–protein crosslinks.

Properties of Purified Lens Transglutaminase and Regulation of its Transamidase/Crosslinking Activity by GTP

On account of its protein crosslinking activity, the Ca2+-dependent transglutaminase of the lens is likely to be involved in the formation of cataracts. We have now purified the rabbit lens enzyme to near homogeneity as judged by SDS-PAGE (Mr∼78 kDa), and a key feature of the procedure was the use of a highly selective affinity chromatographic step with a fibronectin fragment as ligand. The catalytic activity of the lens transglutaminase, measured by the incorporation of dansylcadaverine into dimethylcasein, was compared with those of two similar enzymes isolated from human red cells and from guinea pig liver, respectively. All three enzymes were inhibited by GTP, but the lens enzyme was most sensitive to inhibition by the nucleotide. Moreover, GTP was also shown to inhibit the formation of the ∼55 kDa βcrystallin dimers in the Ca2+-treated rabbit lens homogenate, proving that the nucleotide is a negative regulator for the crosslinking activity of transglutaminase in this tissue.

Kinematic changes following orthopedic surgery in persons status post traumatic brain injury

Two colorimetric assays for tissue transglutaminase (type II) activity involving the crosslinking of proteins have been developed. In one assay, biotin labelled casein is crosslinked into chemically modified casein bound to a microtiter plate by tissue transglutaminase and the biotin labelled reaction product is detected by conjugation to Extravidin peroxidase. The assay can detect activity in 10 ng of commercially available purified guinea pig liver transglutaminase and in the crude homogenate derived from 400 human endothelial cells (cell line ECV 304). A correlation (r2=0.977) was shown between this assay and the radiolabeled putrescine incorporation assay for the detection of transglutaminase activity. This assay measures the protein crosslinking activity of tissue transglutaminase as opposed to polyamine incorporation and offers a rapid, non-radiometric method for screening large sample numbers. Typical inter-assay variability is 13.9±1.5% (n=8). In a second assay, the ability of tissue transglutaminase to catalyze the formation of N′,N′-bis (γ-glutamyl) polyamine bridges is measured. N′,N′-dimethylcasein is bound to a microtiter plate and modified enzymatically using commercially available purified guinea pig liver transglutaminase to incorporate polyamines into glutamine residues. Biotin labelled casein is then crosslinked into the immobilized polyamines by tissue transglutaminase resulting in the formation of N′,N′-bis (γ-glutamyl) polyamine linkages.

Surimi Gel Enhancement by Bovine Plasma Proteins

Gel strength enhancement of Pacific whiting surimi was studied by using an α2-macroglobulin (α2-M)-enriched plasma fraction (FIV-1) and a transglutaminase (TG)-enriched plasma fraction (FI-S). Fluorescent amine-incorporating activity was detected in FIV-1, FI-S, and bovine plasma protein (BPP), indicating potential protein cross-linking activity by α2-M and TG. Inhibition of autolytic activity was observed with FIV-1 and BPP. FIV-1 in combination with bovine serum albumin, fibrinogen, or FI-S enhanced gelation more than FIV-1 alone. These results indicate various components in BPP function both to enhance gelation of Pacific whiting surimi and to inhibit proteolysis. Keywords: Plasma protein; transglutaminase; α2-macroglobulin; surimi; gelation

Assays for the measurement of tissue transglutaminase (type II) mediated protein crosslinking via ϵ-( γ-glutamyl) lysine and N′,N′-bis ( γ-glutamyl) polyamine linkages using biotin labelled casein

Two colorimetric assays for tissue transglutaminase (type II) activity involving the crosslinking of proteins have been developed. In one assay, biotin labelled casein is crosslinked into chemically modified casein bound to a microtiter plate by tissue transglutaminase and the biotin labelled reaction product is detected by conjugation to Extravidin peroxidase. The assay can detect activity in 10 ng of commercially available purified guinea pig liver transglutaminase and in the crude homogenate derived from 400 human endothelial cells (cell line ECV 304). A correlation ( r 2=0.977) was shown between this assay and the radiolabeled putrescine incorporation assay for the detection of transglutaminase activity. This assay measures the protein crosslinking activity of tissue transglutaminase as opposed to polyamine incorporation and offers a rapid, non-radiometric method for screening large sample numbers. Typical inter-assay variability is 13.9±1.5% ( n=8). In a second assay, the ability of tissue transglutaminase to catalyze the formation of N′,N′-bis ( γ-glutamyl) polyamine bridges is measured. N′,N′-dimethylcasein is bound to a microtiter plate and modified enzymatically using commercially available purified guinea pig liver transglutaminase to incorporate polyamines into glutamine residues. Biotin labelled casein is then crosslinked into the immobilized polyamines by tissue transglutaminase resulting in the formation of N′,N′-bis ( γ-glutamyl) polyamine linkages.

ASCORBIC ACID GLYCATION OF LENS PROTEINS PRODUCES UVA SENSITIZERS SIMILAR TO THOSE IN HUMAN LENS

Soluble calf lens proteins were extensively glycated during a 4 week incubation with ascorbic acid in the presence of oxygen. Amino acid analysis of the dialyzed proteins removed at weekly intervals showed an increasing loss of lysine, arginine and histidine, consistent with the extensive protein cross-linking observed. Irradiation of the dialyzed samples with UVA light (1.0 kJ/cm2 total illumination through a 338 nm cutoff filter) caused an increasing loss of tryptophan, an additional loss of histidine and the production of micromolar concentrations of hydrogen peroxide. No alteration in amino acid content and no photolytic effects were seen in proteins incubated without ascorbic acid or in proteins incubated with glucose for 4 weeks. The rate of hydrogen peroxide formation was linear with each glycated sample with a maximum production of 25 nmol/mg protein illuminated. The possibility that the sensitizer activity was due to an ascorbate-induced oxidation of tryptophan was eliminated by the presence of a heavy metal ion chelator during the incubation and by showing equivalent effects with ascorbate-incubated ribonuclease A, which is devoid of tryptophan. The ascorbate-incubated samples displayed increasing absorbance at wavelengths above 300 nm and increasing fluorescence (340/430) as glycation proceeded. The spectra of the 4 week glycated proteins were identical to those obtained with a solubilized water-insoluble fraction from human lens, which is known to have UVA sensitizer activity. The incubation of lens proteins with dehydroascorbic acid or L-threose, but not fructose, produced equivalent glycation, protein crosslinking and sensitizer activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The importance of the GTP-binding protein tissue transglutaminase in the regulation of cell cycle progression

Tissue transglutaminase (tTgase) is a GTP-binding Ca 2+-dependent enzyme which catalyses the post-translational modification of proteins via ϵ(γ-glutamyl) lysine bridges. Recent evidence suggests that the GTP-binding activity of tTgase may be important in intracellular signalling thus explaining some of the diverse suggested roles for the enzyme. In the following work a malignant hamster fibrosarcoma (Met B) has been stably transfected with both the full length tTgase cDNA (wild type) and a mutant form of the cDNA whereby the active site cysteine (Cys 277) has been replaced by serine. Expression of this mutant cDNA leads to a protein with GTP binding activity which is deficient of protein crosslinking activity. When synchronised into S-phase and allowed to progress through the cell cycle tTgase transfected clones (both mutant and wild type), when compared to transfected controls, show a delayed progression from S-phase to G 2/M when analysed by flow cytometry which appears to be elicited by the G-protein activity of the tTgase.

Transglutaminase C in cerebellar granule neurons: regulation and localization of substrate cross-linking.

Covalent cross-linking of cell surface proteins by the calcium-dependent enzyme transglutaminase C may be implicated in cell-cell interactions and growth regulation. We demonstrate the presence of the enzyme in rat cerebellar cortex during postnatal development. Transglutaminase C was induced in cerebellar granule neurons in culture by retinoic acid, dibutyryl- and 8-bromo-cyclic AMP analogues and by cultivation on a biomatrix substratum. Cyclic AMP analogues stimulated transglutaminase activity in protein synthesis-dependent and -independent phases. The enzyme was distributed at focal adhesion sites on the axon. By calcium-dependent covalent incorporation of the primary amine acceptor substrate, 5-(biotinamido)pentylamine, an increase in the Ca(2+)-dependent cross-linking of at least 11 substrate proteins in the presence of retinoic acid and dibutyryl-cyclic AMP was detected. Of these substrates, a subset was labelled on the surface of living granule neurons. A low-molecular-weight substrate, p18, was tentatively identified as the retinoic acid-inducible neurite-promoting factor, midkine. Transglutaminase-mediated amine incorporation, midkine and isopeptide cross-links were co-localized to axonal adhesion sites. The results provide evidence of transglutaminase C-catalysed protein cross-linking activity in cerebellar granule neurons and its possible implication in cell-substratum interactions.

Pyrrolizidine alkaloid-induced DNA-protein cross-links.

Pyrrolizidine alkaloids (PAs) are potent carcinogenic and anti-mitotic compounds produced by a large number of plant species. In this study, we investigated in vitro the DNA-protein cross-linking activity of several structurally diverse PAs. The DNA cross-linked proteins induced by PAs were also isolated and characterized in mammalian cells. At 300 and 500 microM, the pyrrolic PAs (dehydrosenecionine, dehydromonocrotaline, dehydroseneciphylline, dehydroriddelliine) induced potent DNA cross-links. Protein-associated DNA cross-links accounted for approximately 50% of the total cellular DNA cross-links at 300 microM. The simple necine pyrrole dehydroretronecine induced few DNA--protein cross-links and none were detected with indicine N-oxide. The major proteins cross-linked to DNA from either PA-exposed cells or pyrrolic PA-exposed nuclei were in the molecular weight 40-60 kDa range and were primarily acidic in nature (Ca. pI 4.2-5.0). The patterns of the proteins cross-linked to DNA were similar to that induced by standard bifunctional alkylating agents mitomycin C, cisdichlorodiammine platinum(II) and nitrogen mustard. The macrocyclic pyrrole dehydrosenecionine induced DNA cross-links in pBR322 plasmid DNA with BSA as a protein target. Our data indicated that pyrrolic PAs with a macrocyclic diester such as dehydrosenecionine, dehydroseneciphylline, dehydroriddelliine and dehydromonocrotaline were more potent cross-linkers than the simple necine pyrrolic dehydroretronecine. Cross-linking potency of the PAs examined here coincides with known potency differences in animal toxicity and led us to conclude that DNA--protein cross-linking activity is probably involved in PA-related

Activity and distribution of tissue transglutaminase in association with nerve-muscle synapses.

We have measured, characterized, and localized calcium-dependent protein cross-linking activity in rat skeletal muscle, and in myotubes cultured independently or in coculture with spinal neurones, catalyzed by the enzyme tissue transglutaminase (tTG). The enzyme activity was present in both motor endplate and endplate-free zones of rat diaphragm muscle. tTG in the endplate zone was more tightly associated with the tissue. This form of association was absent in extracts of peripheral nerve. Cross-linking of endogenous proteins, as measured by the content of epsilon-(gamma-glutamyl)lysine isopeptide, was higher in the endplate than in the nonendplate zone. Cytosolic (C) and particulate (B) forms of tTG were separated by ion-exchange chromatography from both regions of the muscle. In the motor endplate zone, a higher proportion of tightly bound tTG was recovered as a separate (B1) particulate form. Km values for calcium activation of the three forms of tTG were in the range of 5-15 microM. Immunocytochemistry with polyclonal and monoclonal antibodies revealed the enzyme at motor endplates and at contacts between neurites of rat embryo spinal neurones and myotubes in primary cocultures. Appearance of the B1 transglutaminase could be induced by coculturing myotubes of the mouse C2C12 cell line with neurones. The results suggest that tTG is most concentrated and active at the motor endplate.

Annulin, a protein expressed at limb segment boundaries in the grasshopper embryo, is homologous to protein cross-linking transglutaminases

Annulin, a protein whose general stage- and position-specific expression pattern in the grasshopper embryo is described in the companion paper, is expressed in epithelial annuli in developing limbs. Here, we show that these annuli comprise narrow circumferential bands of epithelial cells at the boundaries of limb segments. At most boundaries, expression of annulin precedes the first morphological signs of segmentation. The most distal cells in a band underlie the boundary invagination. Bands arise in a stereotyped order and, within a band, expression occurs in an ordered circumferential progression. Annulin has a molecular weight of about 97 kDa and appears to be intracellular and peripherally associated with the inner leaflet of the cell membrane. Using the monoclonal antibody 7H7, two overlapping cDNA clones encoding this protein were isolated from an embryonic Schistocerca cDNA expression library. The nucleotide and deduced amino acid sequences indicate that the annulin protein does not contain either a signal sequence or a transmembrane domain. By sequence comparison, annulin appears to be a transglutaminase, one of a family of enzymes that have protein cross-linking activity. Its expression pattern within the limb and the embryo is associated with areas undergoing morphogenetic rearrangements, movements, or rapid cell division. It may stabilize cells under mechanical stress or participate in some other way in these morphogenetic activities.