Characterization of sea urchin transglutaminase, a protein regulated by guanine/adenine nucleotides

Transglutaminases (TGs) are calcium-dependent enzymes that catalyze the transamidation of glutamine residues to form intermolecular isopeptide bonds. Nine distinct TGs have been identified in mammals, and three of them (types 2, 3, and 5) are regulated by GTP/ATP and are able to hydrolyze GTP, working as bifunctional enzymes. We have isolated a cDNA clone encoding a TG from a cDNA library prepared from the blastula stage of sea urchin Paracentrotus lividus (PlTG). The cDNA sequence has an open reading frame coding for a protein of 738 amino acids, including a Cys active site and two other residues critical for catalytic activity, His and Asp. We have studied its expression pattern by in situ hybridization and have also demonstrated that the in vitro expressed PlTG had GTP- and ATP-hydrolyzing activity; moreover, GTP inhibited the transamidating activity of this enzyme as it does that of human TG2, TG3, and TG5.     

Transglutaminase inhibition: A therapy to protect cells from death in neurodegeneration?

Transglutaminases(TGs;E.C.2.3.2.13)are ubiquitous enzymes which catalyze post-translational modifications of proteins.TGs and TG-catalyzed post-translational modifications of proteins have been shown to be involved in the molecular mechanisms responsible for several human diseases.In particular,TG activity has been hypothesized to also be involved also in the molecular mechanisms responsible for human neurodegenerative diseases.In support of this hypothesis,Basso et al recently demonstrated that the TG inhibition protects against oxidative stress-induced neuronal death,suggesting that multiple TG isoforms participate in oxidative stress-induced cell death and that nonselective TG isoform inhibitors will be most effective in fighting oxidative death in neurological disorders.In this commentary,we discuss the possible molecular mechanisms by which TG activity could be involved in the pathogenesis of neurological diseases,with particular reference to neurodegenerative diseases,and the possible involvement of multiple TG isoforms expressed simultaneously in the nervous system in these diseases.Moreover,therapeutic strategies based on the use of selective or nonselective TG inhibitors for the amelioration of thesymptoms of patients with neurological diseases,characterized by aberrant TG activity,are also discussed.     

Transglutaminases and neurodegeneration

Transglutaminases (TGs) are Ca²⁺-dependent enzymes that catalyze a variety of modifications of glutaminyl (Q) residues. In the brain, these modifications include the covalent attachment of a number of amine-bearing compounds, including lysyl (K) residues and polyamines, which serve to either regulate enzyme activity or attach the TG substrates to biological matrices. Aberrant TG activity is thought to contribute to Alzheimer disease, Parkinson disease, Huntington disease, and supranuclear palsy. Strategies designed to interfere with TG activity have some benefit in animal models of Huntington and Parkinson diseases. The following review summarizes the involvement of TGs in neurodegenerative diseases and discusses the possible use of selective inhibitors as therapeutic agents in these diseases.     

Expression of different forms of transglutaminases by immature cells of Helianthus tuberosus sprout apices

Immature cells of etiolated apices of sprouts growing from Helianthus tuberosus (H. t.) tubers showed Ca²⁺-dependent transglutaminase (TG, EC 2.3.2.13) activity on fibronectin (more efficiently) and dimethylcasein as substrates. Three main TG bands of about 85, 75 and 58 kDa were isolated from the 100,000×g apices supernatant through a DEAE-cellulose column at increasing NaCl concentrations and immuno-identified by anti-TG K and anti-rat prostate gland TG antibodies. These three fractions had catalytic activity as catalyzed polyamine conjugation to N-benzyloxycarbonyl-L-γ-glutaminyl-L-leucine (Z-L-Gln-L-Leu) and the corresponding glutamyl-derivatives were identified. The amino acid composition of these TG proteins was compared with those of several sequenced TGs of different origin. The composition of the two larger bands presented great similarities with annotated TGs; in particular, the 75 kDa form was very similar to mammalian inactive EPB42. The 58 kDa form shared a low similarity with other TGs, including a maize sequence of similar molecular mass, which, however, did not present the catalytic triad in the position of all annotated TGs. A 3D model of the H. t. TGs was built adopting TG2 as template. These novel plant TGs are hypothesized to be constitutive and discussed in relation to their possible roles in immature cells. These data suggest that in plants, multiple TG forms are active in the same organ and that plant and animal enzymes probably are very close not only for their catalytic activity but also structurally.     

The plant extracellular transglutaminase: what mammal analogues tell

The extracellular transglutaminases (TGs) in eukaryotes are responsible for the post-translational modification of proteins through different reactions, cross-linking being the best known. In higher plants, extracellular TG appears to be involved in roles similar to those performed by the mammalian counterparties. Since TGs are pleiotropic enzymes, to fully understand the role of plant enzymes it is possible to compare them with animal TGs, the most studied being TG of type 2 (TG2). The extracellular form of TG2 stabilizes the matrix and modulates the interaction of the integrin-fibronectin receptor, causing the adhesion of cells to the extracellular matrix; TG2 plays a role also in the pathogenicity. Extracellular TGs have also been identified in the cell wall of fungi, such as Candida and Saccharomyces, where they cross-link structural glycoproteins, and in Phytophthora, where they are involved in pathogenicity; in the alga Chlamydomonas, TGs link polyamines to glycoproteins thereby favouring the strengthening of cell wall. In higher plants, TG localized in the cell wall of flower petals appears to be involved in the structural reinforcement as well as senescence and cell death of the flower corolla. In the pollen cell wall an extracellular TG co-localizes with substrates and cross-linked products; it is required for the apical growth of pollen tubes. The pollen TG is also secreted into the extracellular matrix possibly allowing the migration of pollen tubes during fertilisation. Although pollen TGs seem to be secreted via vesicles transported along actin filaments, a different mechanism from the classical ER-Golgi pathway is possible, similar to TG2.     

A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis and inflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation betweens spots was 2.6% (n = 3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n = 3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.     

Thematic review series: glycerolipids. DGAT enzymes and triacylglycerol biosynthesis

Triacylglycerols (triglycerides) (TGs) are the major storage molecules of metabolic energy and FAs in most living organisms. Excessive accumulation of TGs, however, is associated with human diseases, such as obesity, diabetes mellitus, and steatohepatitis. The final and the only committed step in the biosynthesis of TGs is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. The genes encoding two DGAT enzymes, DGAT1 and DGAT2, were identified in the past decade, and the use of molecular tools, including mice deficient in either enzyme, has shed light on their functions. Although DGAT enzymes are involved in TG synthesis, they have distinct protein sequences and differ in their biochemical, cellular, and physiological functions. Both enzymes may be useful as therapeutic targets for diseases. Here we review the current knowledge of DGAT enzymes, focusing on new advances since the cloning of their genes, including possible roles in human health and diseases.     

Visualisation of transglutaminase-mediated cross-linking activity in germinating pollen by laser confocal microscopy

Abstract

Transglutaminases (TGs) are a multigenic family of calcium-dependent protein cross-linking enzymes, which are present in animal and plant cells. We have previously reported the presence of TGs in the cytosol and, more recently, in the cell wall of Malus domestica pollen, where it may be involved in pollen germination and pollen–stylar interactions. In this report we describe a simple method for the in situ visualisation of TG activity in germinating pollen. The method is based on the incorporation, mediated by pollen TG, of a fluorescently labelled exogenous diamine substrate of TG (fluorescein-cadaverine) into endogenous pollen substrates. Following the in situ TG activity reaction, the presence of cross-linked pollen proteins was visualised in fixed specimens of germinated pollen by laser confocal microscopy. Our data indicate the presence of TG cross-linking activity mainly at the apical part of the pollen tube, in the region proximal to the grain, and in the pollen grain itself. In planta, the products of this activity may provide strength to the pollen tube migrating through the style.     

Variations in Both TG1 and TG2 Isozyme-specific In Situ Activities and Protein Expressions during Mouse Embryonic Development

Transglutaminase (TG) is a family of enzymes that catalyzes cross-linking reactions among proteins. Using fluorescent-labeled highly reactive substrate peptides, we recently developed a system to visualize isozyme-specific in situ enzymatic activity. In the present study, we investigated the in situ activities of TG1 (skin-type) and TG2 (tissue-type) using whole mouse sections of various embryonic developmental stages and neonates. In each case, we also successfully used immunostaining of identical whole mouse sections for protein expression after detection of enzymatic activities. In general, the enzymatic activity was correlated with TG protein expression. However, in some tissues, TG protein expression patterns, which were inconsistent with the enzymatic activities, suggested that inactive TGs were produced possibly by self cross-linking or other modifications. Our method allowed us to simultaneously observe developmental variations in both TG isozyme-specific activities and protein levels in mouse embryonic and neonate tissues.     

Physio-pathological roles of transglutaminase-catalyzed reactions

Transglutaminases (TGs) are a large family of related and ubiquitous enzymes that catalyze post-translational modifications of proteins. The main activity of these enzymes is the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. In addition to lysyl residues, other second nucleophilic co-substrates may include monoamines or polyamines (to form mono- or bi-substituted /crosslinked adducts) or -OH groups (to form ester linkages). In the absence of co-substrates, the nucleophile may be water, resulting in the net deamidation of the glutaminyl residue. The TG enzymes are also capable of catalyzing other reactions important for cell viability. The distribution and the physiological roles of TG enzymes have been widely studied in numerous cell types and tissues and their roles in several diseases have begun to be identified. "Tissue" TG (TG2), a member of the TG family of enzymes, has definitely been shown to be involved in the molecular mechanisms responsible for a very widespread human pathology: i.e. celiac disease (CD). TG activity has also been hypothesized to be directly involved in the pathogenetic mechanisms responsible for several other human diseases, including neurodegenerative diseases, which are often associated with CD. Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, supranuclear palsy, Huntington's disease and other recently identified polyglutamine diseases, are characterized, in part, by aberrant cerebral TG activity and by increased cross-linked proteins in affected brains. In this review, we discuss the physio-pathological role of TG-catalyzed reactions, with particular interest in the molecular mechanisms that could involve these enzymes in the physio-pathological processes responsible for human neurodegenerative diseases.     

Preferential hydrolysis of monohydroperoxides of linoleoyl and linolenoyl triacylglycerol by pancreatic lipase

Four triacylglycerols (TGs) containing palmitoyl and linoleoyl or linolenoyl groups in known positions were synthesized and pancreatic lipase hydrolysis of their monohydroperoxides was investigated. TG monohydroperoxides did not deactivate the lipase and were hydrolyzed at almost the same degrees as their original TGs. In the hydrolysis of unoxidized TGs, pancreatic lipase showed almost the same reactivity on palmitoyl, linoleoyl and linolenoyl groups at the 1(3)-positions. However, this enzyme had fatty acid specificity for TG monohydroperoxides and the molar concentration of hydroperoxy linoleic or linolenic acid liberated from 1(3)-positions of TG monohydroperoxides were 1.6-2.4-times higher than that of the unoxidized fatty acid from the corresponding 3(1)-positions. The susceptibility of hydroperoxy acyl components of TG monohydroperoxides to pancreatic lipase hydrolysis is explained by its molecular structure and hydrophilic property.     

Characterization of the transglutaminase gene family in zebrafish and in vivo analysis of transglutaminase-dependent bone mineralization

We have characterized the protein cross-linking enzyme transglutaminase (TGs) genes in zebrafish, Danio rerio, based on the analysis of their genomic organization and phylogenetics. Thirteen zebrafish TG genes (zTGs) have been identified, of which 11 show high homology to only 3 mammalian enzymes: TG1, TG2 and FXIIIa. No zebrafish homologues were identified for mammalian TGs 3-7. Real-time PCR analysis demonstrated distinct temporal expression profiles for zTGs in larvae and adult fish. Analysis by in situ hybridization revealed restricted expression of zTG2b and zFXIIIa in skeletal elements, resembling expression of their mammalian homologues in osteo-chondrogenic cells. Mammalian TG2 and FXIIIa have been implicated in promoting osteoblast differentiation and bone mineralization in vitro, however, mouse models lacking either gene have no skeletal phenotype likely due to a compensation effect. We show in this study that mineralization of the newly formed vertebrae is significantly reduced in fish grown for 5?days in the presence of TG inhibitor KCC-009 added at 3–5?days post fertilization. This treatment reduces average vertebrae mineralization by 30%, with complete inhibition in some fish, and no effect on the overall growth and vertebrae number. This is the first in vivo demonstration of the crucial requirement for the TG-catalyzed cross-linking activity in bone mineralization.     

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.     

Transglutaminase 2 as a biomarker of osteoarthritis: an update

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodelling. Under physiologic conditions, articular cartilage displays a stable chondrocyte phenotype, whereas in osteoarthritis a chondrocyte hypertrophy develops near the sites of cartilage surface damage and associates to the pathologic expression of type X collagen. Transglutaminases (TGs) include a family of Ca²⁺-dependent enzymes that catalyze the formation of γ-glutamyl cross-links. Their substrates include a variety of intracellular and extracellular macromolecular components. TGs are ubiquitously and abundantly expressed and implicated in a variety of physiopathological processes. TGs activity is modulated by inflammatory cytokines. TG2 (also known as tissue transglutaminase) mediates the hypertrophic differentiation of joint chondrocytes and interleukin-1-induced calcification. Histomorphometrical and biomolecular investigations document increased TG2 expression in human and experimental osteoarthritis. Consequently, the level of TG2 expression may represent an adjuvant additional marker to monitor tissue remodelling occurring in osteoarthritic joint tissue. Experimental induction of osteoarthritis in TG2 knockout mice is followed from reduced cartilage destruction and increased osteophyte formation compared to wild-type mice, suggesting a different influence on joint bone and cartilage remodelling. The capacity of transamidation by TG2 to regulate activation of latent TGF-β seems to have a potential impact on the regulation of inflammatory response in osteoarthritic tissues. Additional studies are needed to define TG2-regulated pathways that are differently modulated in osteoblasts and chondrocytes during osteoarthritis.     

Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate

Transglutaminase 2 (TG2) is a ubiquitously expressed multifunctional protein with Ca²⁺-dependent transamidase activity forming protease-resistant N^ε-(γ-glutamyl) lysine crosslinks between proteins. It can also function as an isopeptidase cleaving the previously formed crosslinks. The biological significance of this activity has not been revealed yet, mainly because of the lack of a protein-based method for its characterization. Here we report the development of a novel kinetic method for measuring isopeptidase activity of human TG2 by monitoring decrease in the fluorescence polarization of a protein substrate previously formed by crosslinking fluorescently labeled glutamine donor FLpepT26 to S100A4 at a specific lysine residue. The developed method could be applied to test mutant enzymes and compounds that influence isopeptidase activity of TG2.     

A novel transglutaminase-mediated post-translational modification of phospholipase A2 dramatically increases its catalytic activity

Transglutaminases (TG), which include coagulation Factor XIIIa, are calcium-dependent ubiquitous enzymes. TGs catalyze the formation of an isopeptide bond by cross-linking a specific glutamine and a lysine residue between two proteins or within the same protein molecule. Phospholipase A2 (PLA2) is a key enzyme in the regulation of prostaglandin and leukotriene biosynthetic pathways, which catalyzes the release of free fatty acids from the sn-2 position of membrane glycerophospholipids. This enzyme has been suggested to be pathophysiologically related to the initiation and propagation of several inflammatory diseases including juvenile rheumatoid and rheumatoid arthritis. Here, we describe a novel TG-catalyzed post-translational modification of PLA2 which dramatically increases the activity of this enzyme. This increase was dependent upon the time of preincubation, the concentration of TG and the presence of Ca2+. Size exclusion chromatography of TG-treated PLA2 yielded two peaks of PLA2 activity, with apparent molecular masses of 26 and 13 kDa, respectively. The 26-kDa species, a putative PLA2 dimer, contained epsilon-(gamma-glutamyl)-lysine isopeptide in about 1:1 molar ratio to PLA2, suggesting an intramolecular rather than intermolecular cross-linking. This hypothesis was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the 26- and 13-kDa species under denaturing conditions. The specific activity of the dimeric peak was 10-fold higher with respect to that of the monomeric enzyme. These data suggest that TG-catalyzed covalent cross-linking of PLA2 is intramolecular and that this may promote a noncovalent dimerization and subsequent activation of this enzyme via a conformational change. To our knowledge, this is the first demonstration that TG-mediated post-translational modification of an enzyme (e.g. PLA2) causes a striking increase in the catalytic activity of the enzyme.     

Triacylglycerol hydrolase is localized to the endoplasmic reticulum by an unusual retrieval sequence where it participates in VLDL assembly without utilizing VLDL lipids as substrates

The majority of hepatic intracellular triacylglycerol (TG) is mobilized by lipolysis followed by reesterification to reassemble TG before incorporation into a very-low-density lipoprotein (VLDL) particle. Triacylglycerol hydrolase (TGH) is a lipase that hydrolyzes TG within hepatocytes. Immunogold electron microscopy in transfected cells revealed a disparate distribution of this enzyme within the endoplasmic reticulum (ER), with particularly intense localization in regions surrounding mitochondria. TGH is localized to the lumen of the ER by the C-terminal tetrapeptide sequence HIEL functioning as an ER retention signal. Deletion of HIEL resulted in secretion of catalytically active TGH. Mutation of HIEL to KDEL, which is the consensus ER retrieval sequence in animal cells, also resulted in ER retention and conservation of lipolytic activity. However, KDEL-TGH was not as efficient at mobilizing lipids for VLDL secretion and exhibited an altered distribution within the ER. TGH is a glycoprotein, but glycosylation is not required for catalytic activity. TGH does not hydrolyze apolipoprotein B-associated lipids. This suggests a mechanism for vectored movement of TGs onto developing VLDL in the ER as TGH may mobilize TG for VLDL assembly, but will not access this lipid once it is associated with VLDL.     

Transglutaminase activity regulates osteoblast differentiation and matrix mineralization in MC3T3-E1 osteoblast cultures.

Transglutaminase (TG) enzymes and protein crosslinking have long been implicated in the formation of mineralized tissues. The aim of this study was to analyze the expression, activity and function of TGs in differentiating osteoblasts to gain further insight into the role of extracellular matrix protein crosslinking in bone formation. MC3T3-E1 (subclone 14) pre-osteoblast cultures were treated with ascorbic acid and beta-glycerophosphate to induce cell differentiation and matrix mineralization. Expression of TG isoforms was analyzed by RT-PCR. TG activity was assessed during osteoblast differentiation by in vitro biochemical assays and by in situ labeling of live cell cultures. We demonstrate that MC3T3-E1/C14 osteoblasts express two TG isoforms--TG2 and FXIIIA. Abundant TG activity was observed during cell differentiation which increased significantly after thrombin treatment, a result confirming the presence of FXIIIA in the cultures. Ascorbic acid treatment, which stimulated collagen secretion and assembly, also stimulated externalization of TG activity, likely from FXIIIA which was externalized upon this treatment as analyzed by immunofluoresence microscopy. Inhibition of TG activity in the cultures by cystamine resulted in complete abrogation of mineralization, attributable to decreased matrix accumulation and an arrested state of osteoblast differentiation as measured by decreased levels of bone sialoprotein, osteocalcin and alkaline phosphatase. Additional functional studies and substrate characterization showed that TG activity was required for the formation of a fibronectin-collagen network during the early stages of matrix formation and assembly. This network, in turn, appeared to be essential for further matrix production and progression of the osteoblast differentiation program, and ultimately for mineralization.