Analysis of transglutaminase protein substrates by functional proteomics

Transglutaminases are calcium-dependent enzymes that catalyze a post-translational modification of proteins through the formation of epsilon -(gamma-glutamyl)lysine bonds. Although specific roles for transglutaminases have been described, recent findings have provided evidence that dysregulation of transglutaminases may contribute to many pathological processes including celiac disease and neurodegenerative diseases. A crucial step in the elucidation of biological and pathological roles of transglutaminases requires the identification of protein substrates. A strategy based on a functional proteomic analysis was set up using two well-characterized biotinylated transglutaminase substrates as affinity probes: 5-(biotinamido)pentylamine and the synthetic biotinylated peptide TVQQEL, the amino- and acyl-donor probes, respectively. A pool of known tissue type transglutaminase protein substrates was selected in order to test the procedure. Results obtained in this paper indicate that the whole strategy can be successfully applied in order to identify transglutaminases protein substrates as well as the amino acid site sensitive toward enzyme activity.     

Producing transglutaminases by molecular farming in plants: Minireview article

Summary. Transglutaminases have a range of catalytic activities, most of which concern the post-translational modification of proteins. The most important of these activities, both in terms of biology and biotechnology, is the cross-linking of proteins into large supramolecular networks. The widespread use of transglutaminases in research, medicine and industry has increased the demand for an inexpensive, efficient and safe source of recombinant enzymes. We describe initial results concerning the production of a mammalian transglutaminase in transgenic rice plants as a first step towards the large-scale molecular farming of this enzyme.     

Possible involvement of transglutaminase-catalyzed reactions in the physiopathology of neurodegenerative diseases

Transglutaminases are ubiquitous enzymes, which catalyze post-translational modifications of proteins. Recently, transglutaminases and tranglutaminase-catalyzed post-translational modification of proteins have been shown to be involved in the molecular mechanisms responsible for several human diseases. Transglutaminase activity has been hypothesized to be involved also in the pathogenetic mechanisms responsible for human neurodegenerative diseases. Neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, supranuclear palsy, Huntington’s disease and other polyglutamine diseases, are characterized in part by aberrant cerebral transglutaminase activity and by increased cross-linked proteins in affected brains. In this review, we focus on the possible molecular mechanisms by which transglutaminase activity could be involved in the pathogenesis of neurodegenerative diseases, and on the possible therapeutic effects of selective transglutaminase inhibitors for the cure of patients with diseases characterized by aberrant transglutaminase activity.     

Activation of transglutaminase in mu-calpain null erythrocytes

Intracellular transglutaminases (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) are calcium-dependent thiol enzymes that catalyze the covalent cross-linking of proteins, including those in the erythrocyte membrane. Several studies suggest that the activation of some transglutaminases is positively regulated by the calcium-dependent cysteine protease, mu-calpain. Using mu-calpain null (Capn1(-/-)) mouse erythrocytes, we demonstrate that the activation of soluble as well as membrane-bound forms of transglutaminase (TG2) in mouse erythrocytes was independent of mu-calpain. Also, the absence of mu-calpain or any detectable cysteine protease did not affect the transglutaminase activity in the erythrocyte lysate. Our studies also identify physiological substrates of mu-calpain in the erythrocyte membrane and show that their cleavage has no discernible effect on the transglutaminase mediated cross-linking of membrane proteins. Taken together, these data suggest the existence of a calpain-independent mechanism for the activation of transglutaminase 2 by calcium ions in the mouse erythrocytes and presumably also in non-erythroid cells.     

Expression in Escherichia coli and purification of hexahistidine-tagged human tissue transglutaminase

Recent evidence suggests that aberrant transglutaminase activity is associated with a wide variety of diseases. Tissue transglutaminase is the most widely distributed of the six well-characterized transglutaminases in humans. We describe a method for expressing hexahistidine-tagged human tissue transglutaminase in Escherichia coli BL21(DE3) using the pET-30 Ek/LIC expression vector. Purification of the expressed enzyme from suspensions of E. coli cells treated with CelLytic B Bacterial Cell Lysis/Extraction Reagent was accomplished by immobilized metal (Ni2+) affinity column chromatography. The procedure typically yields highly purified and highly active recombinant human tissue transglutaminase in about 1 day (about 0.6 mg/from a 1-liter culture).     

Isolation and characterization of cDNA clones to mouse macrophage and human endothelial cell tissue transglutaminases.

The deduced amino acid sequences for tissue transglutaminases from human endothelial cells and mouse macrophages have been derived from cloned cDNAs. Northern blot analysis of both tissue transglutaminases shows a message size of approximately 3.6-3.7 kilobases. The molecular weights calculated from the deduced amino acid sequences were 77,253 for human endothelial tissue transglutaminase and 76,699 for mouse macrophage tissue transglutaminase. The deduced amino acid sequence for the human endothelial transglutaminase was confirmed by comparison with the amino acid sequence obtained by cyanogen bromide digestion of the human erythrocyte transglutaminase. The amino acid sequences of both human endothelial and mouse macrophage tissue transglutaminases were compared to other transglutaminases. A very high degree of homology was found between human endothelial, mouse macrophage, and guinea pig liver tissue transglutaminase (greater than 80%). Moreover, human endothelial tissue transglutaminase was compared with human Factor XIIIa and a very high degree of homology (75% identity) was found in the active site region.     

Structural elements responsible for transglutaminase activity of protein disulphide isomerases and thioredoxins

According to recent results both protein disulphide isomerase (PDI) and thioredoxin (Trx) enzymes have transglutaminase activity which can be linked to the thioredoxin box found in these proteins. Analysis of known protein disulphide isomerase and thioredoxin sequences has revealed the presence of conserved Cys, His and Asp residues required for transglutaminases to catalyze the incorporation of primary amines into protein-bound glutamine residues. The available 3D structures of PDIs and Trxs show that these residues are in close proximity to achieve transglutamylation of substrate proteins. The shared activities of the members of the large protein disulphide isomerase, thioredoxin and transglutaminase enzyme families reviewed here may have general biological significance in the regulation of cellular and tissue processes.     

Human inter-α-inhibitor is a substrate for factor XIIIa and tissue transglutaminase

In this study, we show that inter-α-inhibitor is a substrate for both factor XIIIa and tissue transglutaminase. These enzymes catalyze the incorporation of dansylcadaverine and biotin-pentylamine, revealing that inter-α-inhibitor contains reactive Gln residues within all three subunits. These findings suggest that transglutaminases catalyze the covalent conjugation of inter-α-inhibitor to other proteins. This was demonstrated by the cross-linking between inter-α-inhibitor and fibrinogen by either factor XIIIa or tissue transglutaminase. Finally, using quantitative mass spectrometry, we show that inter-α-inhibitor is cross-linked to the fibrin clot in a 1:20 ratio relative to the known factor XIIIa substrate α2-antiplasmin. This interaction may protect fibrin or other Lys-donating proteins from adventitious proteolysis by increasing the local concentration of bikunin. In addition, the reaction may influence the TSG-6/heavy Chain 2-mediated transfer of heavy chains observed during inflammation.     

Screening of substrate peptide sequences for tissue-type transglutaminase (TGase 2) using T7 phage cDNA library

Transglutaminase (TGase) is a family of enzymes that catalyzes cross-linking reaction between glutamine- and lysine residue of substrate proteins in several mammalian biological events. Substrate proteins for TGase and their physiological relevance have been still in research, continuously expanding. In this study, we have established a novel screening system that enables identification of cDNA sequence encoding favorable primary structure as a substrate for tissue-type transglutaminase (TGase 2), a multifunctional and ubiquitously expressing isozyme. By the screening, we identified several T7 phage clones that displayed substrate peptides for TGase 2 as a translated product from human brain cDNA library. Among the selected clones, the C-terminal region of IKAP, IkappaB kinase complex associated protein, appeared as a highly reactive substrate sequence for TGase 2. This system will open possibility of rapid identification of substrate sequences for transglutaminases at a genetic level.     

Transglutaminase activity of human ER-60

Human recombinant ER-60 was confirmed to have transglutaminase activity by a microtiter plate assay. Transglutaminase activity of ER-60 did not require calcium and was inhibited by cystamine, a substrate analogue. In addition, the transglutaminase activity of ER-60 was not inhibited by SH-blocking reagents. These results suggest that the properties of the transglutaminase activity of ER-60 are different from those in the cases of known mammalian transglutaminases of which the active site includes a cysteine residue.     

Transglutaminases: widespread cross-linking enzymes in plants

BACKGROUND: Transglutaminases have been studied in plants since 1987 in investigations aimed at interpreting some of the molecular mechanisms by which polyamines affect growth and differentiation. Transglutaminases are a widely distributed enzyme family catalysing a myriad of biological reactions in animals. In plants, the post-translational modification of proteins by polyamines forming inter- or intra-molecular cross-links has been the main transglutaminase reaction studied. CHARACTERISTICS OF PLANT TRANSGLUTAMINASES: The few plant transglutaminases sequenced so far have little sequence homology with the best-known animal enzymes, except for the catalytic triad; however, they share a possible structural homology. Proofs of their catalytic activity are: (a) their ability to produce glutamyl-polyamine derivatives; (b) their recognition by animal transglutaminase antibodies; and (c) biochemical features such as calcium-dependency, etc. However, many of their fundamental biochemical and physiological properties still remain elusive. TRANSGLUTAMINASE ACTIVITY IS UBIQUITOUS: It has been detected in algae and in angiosperms in different organs and sub-cellular compartments, chloroplasts being the best-studied organelles. POSSIBLE ROLES: Possible roles concern the structural modification of specific protein substrates. In chloroplasts, transglutaminases appear to stabilize the photosynthetic complexes and Rubisco, being regulated by light and other factors, and possibly exerting a positive effect on photosynthesis and photo-protection. In the cytosol, they modify cytoskeletal proteins. Preliminary reports suggest an involvement in the cell wall construction/organization. Other roles appear to be related to fertilization, abiotic and biotic stresses, senescence and programmed cell death, including the hypersensitive reaction. CONCLUSIONS: The widespread occurrence of transglutaminases activity in all organs and cell compartments studied suggests a relevance for their still incompletely defined physiological roles. At present, it is not possible to classify this enzyme family in plants owing to the scarcity of information on genes encoding them.     

Transglutaminases expression in human supraspinatus tendon ruptures and in mouse tendons

The ethiopathogenesis of rotator cuff disease remains poorly understood. Many studies advocate the importance of extra cellular matrix for the homeostasis of connective tissue. Transglutaminase enzymes family has been studied in the context of connective tissue formation and stabilisation. Here, we investigated transglutaminases expression pattern in biopsies of normal and injured supraspinatus tendons of human shoulders and in the Achilles tendons of transglutaminase 2 knock-out and wild-type mice. Our results show that different transglutaminase family members are differentially expressed in human and mouse tendons, and that transglutaminase 2 is down-regulated at mRNA and protein levels upon human supraspinatus tendon ruptures.     

Immuno-cross reactivity of transglutaminase and cornification marker proteins in the epidermis of vertebrates suggests common processes of soft cornification across species

In differentiating mammalian keratinocytes proteins are linked to the plasma membrane by epidermal transglutaminases through N-epsilon-(gamma-glutamyl)-lysine isopeptide bonds to form the cornified cell envelope. The presence of transglutaminases and their protein substrates in the epidermis of nonmammalian vertebrates is not known. The present study analyses the presence and localization of the above proteins in the epidermis using immuno-cross reactivity across different classes of amniotes. After immunoblotting, some protein bands appear labelled for loricrin, sciellin, and transglutaminase in most species. These proteins are scarce to absent in the epidermis of aquatic species (goldfish and newt) where a stratum corneum is absent or very thin. The molecular weight of transglutaminase immunoreactive bands generally varies between 40 to 62 kDa, with the most represented bands at 52-57 kDa in most species. The more intense loricrin- and sciellin-immunoreactive bands are seen at 50-55-62 kDa, but are weak or absent in aquatic vertebrates. Loricrine-like immunoreactivity is present in the epidermis where alpha-(soft)-keratinization occurs. Isopeptide bonds are mainly associated to bands in the range of 50-62 kDa. In vertebrates where hard-keratin is expressed (the beta-keratin corneous layer of sauropsids and in feathers) or in hair cortex of mammals, no loricrin-like, transglutaminase-, and isopeptide-bond-immunoreactivities are seen. Immunoblotting however shows loricrin-, sciellin-, and trasnsglutaminase-positive bands in the corneous layers containing beta-keratin. Histologically, the epidermis of most amniotes shows variable transglutaminase immunoreactivity, but isopeptide-bond and sciellin immunoreactivities are weak or undetactable in most species. The limitations of immunohistochemical methods are discussed and compared with results from immunoblotting. In reptilian epidermis transglutaminase is mainly localized in 0.15-0.3 microm dense granules or diffuse in transitional alpha-keratogenic cells. In beta-keratogenic cells few small dense granules show a weak immunolabeling. Transglutaminase is present in nuclei of terminal differentiating alpha- and beta-keratinocytes, as in those of mature inner and outer root sheath. The present study suggests that keratinization based on loricrin, sciellin and transglutaminase was probably present in the stratum corneoum of basic amniotes in the Carboniferous. These proteins were mainly maintained in alpha-keratogenic layers of amniotes but decreased in beta-keratogenic layers of sauropsids (reptiles and birds). The study suggests that similar proteins for the formation of the cornified cell envelope are present in alpha-keratinocytes across vertebrates but not in beta-keratinocytes.     

Regulation of the activities of the mammalian transglutaminase family of enzymes

Mammalian transglutaminases catalyze post‐translational modifications of glutamine residues on proteins and peptides through transamidation or deamidation reactions. Their catalytic mechanism resembles that of cysteine proteases. In virtually every case, their enzymatic activity is modulated by elaborate strategies including controlled gene expression, allostery, covalent modification, and proteolysis. In this review, we focus on our current knowledge of post‐translational regulation of transglutaminase activity by physiological as well as synthetic allosteric agents. Our discussion will primarily focus on transglutaminase 2, but will also compare and contrast its regulation with Factor XIIIa as well as transglutaminases 1 and 3. Potential structure–function relationships of known mutations in human transglutaminases are analyzed.     

Rheological characterization and dissolution kinetics of fibrin gels crosslinked by a microbial transglutaminase

Various fibrin gels were prepared with a microbial transglutaminase under miscellaneous conditions. The gels were characterized through their rheological properties. The influence of fibronectin addition and that of covalent bonding on the viscoelastic characteristics were evaluated. Gel elasticity is proportional to fibrinogen concentration but shows a nonlinear dependence on transglutaminase concentration. Additional crosslink of fibronectin in fibrin gels has no effect on the rheological character of the matrix. Dissolution kinetics in concentrated urea solutions evidences the role of covalent bonds on gel stability. The rheological properties and gel stability are discussed in relation with the enzyme-catalyzed covalent bonding. The microbial enzyme reactions are compared to those of FXIII and tissue transglutaminases.     

Secretory leukocyte protease inhibitor (SLPI) is, like its homologue trappin-2 (pre-elafin), a transglutaminase substrate

Human lungs contain secretory leukocyte protease inhibitor (SLPI), elafin and its biologically active precursor trappin-2 (pre-elafin). These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to elafin, but it lacks consensus transglutaminase sequence(s), unlike trappin-2 and elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases.     

Epidermal and hair follicle trans glutaminases and crosslinking in skin

Summary Epidermal and hair follicle transglutaminases crosslink structural proteins in the skin by epsilon-(gamma-glutamyl)-lysine bonds. This crosslinking produces protein polymers that are extremely insoluble and, until recently, difficult to characterize.Epidermal transglutaminase is localized to the granular layer of the epidermis. It catalyzes the crosslinking of a soluble cytoplasmic precursor to form the cornified envelope that lines the inner membrane of the mature keratinocyte in the stratum corneum.Hair follicle transglutaminase is localized to the inner root sheath and medulla of the hair follicle. It crosslinks a poorly characterized citrulline-rich protein.The enzymes and their substrates have been shown to be important markers of normal differentiation. Regulation of these processes is currently under investigation.     

3′-Hydroxy T-2 and 3′-Hydroxy HT-2 Toxins: New Metabolites of T-2 Toxin,a Trichothecene Mycotoxin,in Animals

A microorganism producing transglutaminase was screened as an indication of hydroxamate- forming activity. The microbial transglutaminase was purified from the culture filtrate of the strain, S- 8112, which was supposed to belong to the genus Streptoverticillium. The molecular weight of the purified enzyme was found to be about 40,000 on SDS-polyacrylamide gel electrophoresis, the isoelectric point 8.9 and the optimal pH of the reaction 6~7. The present enzyme requires no calcium ions for its activity. Thus, it clearly differs from known transglutaminases derived from mammalian organs, which have been defined as calcium-dependent enzymes.     

Epidermal type I transglutaminase (TGM1) is assigned to human chromosome 14.

Human epidermal type I transglutaminase coexists in keratinocytes with another cross-linking enzyme, tissue type II transglutaminase. There are at least five different forms of the enzyme in mammals. Gene mapping studies allowed us to determine whether the different transglutaminases are products of the same gene or separate genes. The gene encoding factor XIII subunit a transglutaminase (F13A1) was previously assigned to human chromosome 6, p24----p25. We demonstrate using somatic cell hybrids that the human epidermal type I transglutaminase gene (gene symbol is designated TGM1) is located on human chromosome 14, providing evidence that at least two human transglutaminases are encoded by separate genes.     

Identification of protein substrates for transglutaminase in Caenorhabditis elegans

Transglutaminase-dependent cross-linking of proteins leads to protein polymerisation that confers stability as well as resistance to mechanical disruption and chemical attack. Various transglutaminases have been implicated in a wide range of biological phenomena occurring in both extracellular and intracellular compartments, but further clarification of the physiological role of these enzymes requires identification of possible substrate molecules. Here we report the detection, purification, and identification of two proteins, enolase and ATP synthase alpha subunit as glutamine donor protein substrates for the transglutaminase of the nematode Caenorhabditis elegans.