Transglutaminase Reaction Research Articles

Pir Proteins of Saccharomyces cerevisiae Are Attached to β-1,3-Glucan by a New Protein-Carbohydrate Linkage

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the gamma-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of beta-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.

Influence of weak and covalent bonds on formation and hydrolysis of gelatin networks.

The relative influence of physical and chemical bonds to overall gel properties are explored in gelatin gels. Physical, chemical, chemical-physical, and physical-chemical gels are obtained by cooling the protein solution and/or by transglutaminase reaction. Each type of network is characterized by rheology and polarimetry. It is shown that the overall properties as well as the dynamics inside the gels are dependent upon the order of formation and on the relative amount of triple helices and covalent bonds. Enzyme hydrolysis of covalent gels is slower than that of physical gels, as confirmed by the kinetics of helix release and degradation. A scheme is proposed to explain the results at both the physicochemical and the molecular levels.

In vitro modification of betaine-homocysteine S-methyltransferase by tissue-type transglutaminase

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. To elucidate the physiological roles of transglutaminase at the molecular level, we need to identify its physiological protein substrates and clarify the relationship between transglutaminase modification of protein substrates and biological responses. Here we examined whether betaine-homocysteine S-methyltransferase (BHMT: EC 2.1.1.5) can be a substrate of tissue-type transglutaminase by in vitro experiments using porcine liver BHMT and guinea pig liver transglutarninase. Guinea pig liver transglutaminase incorporated 5-(biotinamido) pentylamine and [ 3 H] histamine into BHMT in a time-dependent manner. Putrescine and spermidine also seemed to be incorporated into BHMT by transglutaminase. In the absence of the primary amines, BHMT subunits were cross-linked intra- and intermolecularly. BHMT activity was decreased significantly through the cross-linking by transglutaminase. Histamine incorporation slightly reduced the BHMT activity. Peptide fragments of BHMT containing the glutamine residues reactive for transglutaminase reaction were isolated through biotin labelling, proteinase digestion, biotin-avidin a affinity separation, and reverse phase HPLC. The results of amino acid sequence analyses of these peptides and sequence homology alignment with other mammalian liver BHMT subunits showed that these reactive glutamine residues were located in the region near the carboxyl terminal of porcine BHMT subunit. These results suggested that the liver BHMT can be modified by tissue-type transglutaminase and its activity is regulated repressively by the modification, especially by the cross-linking. This regulatory reaction might be involved in the regulation of homocysteine metabolism in the liver.

Cystamine inhibits transglutaminase and caspase-3 cleavage in glutamate-exposed astroglial cells.

Although the precise role of transglutaminase in cell death is unknown, several findings demonstrate that tissue transglutaminase selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Calcium-dependent transglutaminase reactions are also implicated in several neurodegenerative diseases, including alterations in the release of excitatory amino acids. One prevalent theme in cell damage induced by excitotoxic stimuli in different regions of the CNS is that apoptosis may be executed by intracellular caspase proteases. Furthermore, the presence of functional ion channel-gated receptors in glial cells suggests that also astrocytes can be susceptible to glutamate's toxic effects. In this study, we demonstrated that prolonged exposure to glutamate (100 microM) of cultured astrocytes caused an increase in the expression of tissue transglutaminase (tTG). This effect was prevented by preincubation with GYKI 52466, an antagonist of AMPA/KA receptors. Glutamate exposure also promoted an increase in caspase-3 compared with control cultures. Confocal laser microscopy analysis demonstrated the presence of activated caspase-3 in the cytoplasm as well as in the nucleus. The inhibition of TG-catalyzed reactions by cystamine (1 mM) blocked the activation pathway of caspase-3, with an evident reduction of enzyme cleavage. These results suggest that glutamate increased both TG and caspase-3 in astroglial cells early in the excitotoxin-induced events.

A novel function of tissue-type transglutaminase: protein disulphide isomerase.

We have found that tissue-type transglutaminase (tTG), also called TGc, TGase2 and Galpha(h), has the activity of protein disulphide isomerase (PDI). We have shown that tTG converts completely reduced/denatured inactive RNase A molecule to the native active enzyme. The PDI activity of tTG was strongly inhibited by bacitracin, which is a frequently used inhibitor of conventional PDI activity. It was substantially inhibited by the simultaneous presence of other potential substrate proteins such as completely reduced BSA, but not by native BSA. This activity was especially high in the presence of GSSG, but not GSH. The addition of GSH to the reaction mixture in the presence of GSSG at a fixed concentration up to at least 200-fold excess did not very substantially inhibit the PDI activity. It is possible that tTG can exert PDI activity in a fairly reducing environment like cytosol, where most of tTG is found. It is quite obvious from the following observations that PDI activity of tTG is catalysed by a domain different from that used for the transglutaminase reaction. Although the alkylation of Cys residues in tTG completely abolished the transglutaminase activity, as was expected, it did not affect the PDI activity at all. This PDI activity did not require the presence of Ca(2+). It was not inhibited by nucleotides including GTP at all, unlike the other activity of tTG.

Excitotoxin-induced changes in transglutaminase during differentiation of cerebellar granule cells.

Excitotoxicity induced by NMDA receptor stimulation is able to increase the activity of many enzymes involved in neuronal cell death. Primary cultures of rat cerebellar granule cells were used to elucidate the role of transglutaminase reaction in the excitotoxic cell response, and to evaluate the role of glutamate receptors in cell survival and degeneration. Granule neurons, maintained in vitro for two weeks, were exposed to NMDA at different stages of differentiation. Following NMDA receptor activation, increases in transglutaminase activity were observed in cell cultures. The levels of enzyme activity were higher in cells at 5 days in vitro than in those at 8-9 or 13-14 days in vitro. Moreover, NMDA exposure up-regulated tTG expression in neurons as young as 5 days in vitro. These cultures also exhibited morphological changes with clear apoptotic features. Results obtained demonstrate that susceptibility of granule cells to excitotoxicity depends on the developmental stage of neurons.

Peptide-Retargeted Adenovirus Encoding a Tissue Inhibitor of Metalloproteinase-1 Decreases Restenosis after Intravascular Gene Transfer

In this study we have attached cyclic targeting peptides by way of a poly-lysine spacer on the surface of an adenovirus using a transglutaminase enzymatic reaction to enhance transduction efficiency and to modify tissue tropism in vivo. Nuclear targeted lacZ- and TIMP-1-encoding adenoviruses were coupled to a peptide-motif (HWGF) that can bind to matrix metalloproteinase (MMP)-2 and MMP-9. Modified viruses were used to evaluate gene transfer efficiency, biodistribution, and the effect on neointima formation following balloon denudation injury. In vitro, both rabbit aortic smooth muscle cells and human endothelial hybridoma cells demonstrated significantly increased reporter gene expression with HWGF-modified adenoviruses (AdlacZ(HWGF)) compared with control (AdlacZ) or mismatch peptide-modified (AdlacZ(MM)) adenoviruses. However, in human hepatocellular Hep-G2 cells, both AdlacZ(HWGF) and AdlacZ(MM) produced significantly lower transgene expression compared with the respective control viruses. In vivo, local intravascular catheter-mediated gene transfer of a HWGF-targeted TIMP-1-encoding adenovirus (AdTIMP-1(HWGF)) significantly reduced intimal thickening in a rabbit aortic balloon denudation model (P < 0.05) compared with the control adenovirus. X-Gal staining and biodistribution analyses with TaqMan RT-PCR revealed that the cyclic peptides altered vector tropism and, in particular, reduced transduction of the liver. We found that the HWGF peptide modification increased transduction efficiency of the adenovirus-mediated gene transfer in smooth muscle cells and endothelial cells in in vitro and enhanced gene transfer to the arterial wall in vivo; that peptide modification of adenoviruses beneficially modulated tissue tropism in vivo; and that efficient TIMP-1 gene transfer reduced intimal thickening in an established restenosis model in rabbits.

CO-OVEREXPRESSION OF FOLDING MODULATORS IMPROVES THE SOLUBILITY OF THE RECOMBINANT GUINEA PIG LIVER TRANSGLUTAMINASE EXPRESSED IN ESCHERICHIA COLI

ABSTRACT Transglutaminases (EC 2.3.2.13) catalyze the formation of ε-(γ-glutamyl)lysine cross-links and the substitution of primary amines for the γ-carboxamide groups of protein bound glutamine residues, and are involved in many biological phenomena. Transglutaminase reactions are also applicable in applied enzymology. Here, we established an expression system of recombinant mammalian tissue-type transglutaminase with high productivity. Overexpression of guinea pig liver transglutaminase in Escherichia coli, using a plasmid pET21-d, mostly resulted in the accumulation of insoluble and inactive enzyme protein. By the expression culture at lower temperatures (25 and 18°C), however, a fraction of the soluble and active enzyme protein slightly increased. Co-overexpression of a molecular chaperone system (DnaK-DnaJ-GrpE) and/or a folding catalyst (trigger factor) improved the solubility of the recombinant enzyme produced in E. coli cells. The specific activity, the affinity to the amine substrate, and the sensitivity to the calcium activation and GTP inhibition of the purified soluble recombinant enzyme were lower than those of the natural liver enzyme. These results indicated that co-overexpression of folding modulators tested improved the solubility of the overproduced recombinant mammalian tissue-type transglutaminase, but the catalytic properties of the soluble recombinant enzyme were not exactly the same as those of the natural enzyme.

Susceptibility of an industrial α-lactalbumin concentrate to cross-linking by microbial transglutaminase

Abstract The susceptibility of an industrial α -lactalbumin concentrate to cross-linking with a microbial transglutaminase from Streptoverticillium mobaraense was investigated. At a protein concentration of 0.5% w v −1 , the maximum cross-linking was observed at 50°C, pH 5 and at 5 h of incubation time. Results from sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis showed that most of the monomeric form of α -lactalbumin was converted to polymers too large to move into the gel matrix. Addition of ethylenediamine tetraacetic acid or SDS prior to the incubation of protein–enzyme mixture, further enhanced the transglutaminase reaction with the industrial α -lactalbumin. Results from reverse phase chromatography indicated that cross-linking caused a broadening of the α -lactalbumin peak with little change in the average hydrophobicity of the protein. In contrast to the reported results on pure α -lactalbumin, the industrial α -lactalbumin concentrate showed considerable cross-linking with transglutaminase even without the reduction of the disulphide bonds. This difference was attributed to the partially unfolded secondary structures in the industrial α -lactalbumin concentrate.

N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL) is increased in cerebrospinal fluid of patients with Huntington's disease.

Pathological-length polyglutamine (Q(n)) expansions, such as those that occur in the huntingtin protein (htt) in Huntington's disease (HD), are excellent substrates for tissue transglutaminase in vitro, and transglutaminase activity is increased in post-mortem HD brain. However, direct evidence for the participation of tissue transglutaminase (or other transglutaminases) in HD patients in vivo is scarce. We now report that levels of N(epsilon)-(gamma-L-glutamyl)-L-lysine (GGEL)--a 'marker' isodipeptide produced by the transglutaminase reaction--are elevated in the CSF of HD patients (708 +/- 41 pmol/mL, SEM, n = 36) vs. control CSF (228 +/- 36, n = 27); p < 0.0001. These data support the hypothesis that transglutaminase activity is increased in HD brain in vivo.

Posttranslational modifications of microfibril associated glycoprotein-1 (MAGP-1).

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight protein associated with extracellular matrix microfibrils. Biochemical studies have shown that MAGP-1 undergoes several posttranslational modifications that may influence its associations with other microfibrillar components. To identify the sites in the molecule where posttranslational modifications occur, we expressed MAGP-1 constructs containing various point mutations as well as front and back half truncations in CHO cells. Characterization of transiently expressed protein showed that MAGP-1 undergoes O-linked glycosylation and tyrosine sulfation at sites in its amino-terminal half. This region of the protein also served as a major amine acceptor site for transglutaminase and mediated self-assembly into high molecular weight multimers through a glutamine-rich sequence. Fine mapping of the modification sites through mutational analysis demonstrated that Gln20 is a major amine acceptor site for the transglutaminase reaction and confirmed that a canonical tyrosine sulfation consensus sequence is the site of MAGP-1 sulfation. Our results also show that O-glycosylation occurs at more than one site in the molecule.

Conformational changes in the nucleosome followed by the selective accessibility of histone glutamines in the transglutaminase reaction: effects of ionic strength.

Transglutaminases, the enzymes that catalyze the acyl-transfer reaction between glutamine and primary amines, have been used to introduce probes into proteins in order to perform structural studies using physical techniques. Here we use an original approach in which the increasing accessibility of the glutamines of core histones to TGase is used to monitor the salt-induced conformational changes of the nucleosome. The rationale of this strategy is that the accessibility of a glutamine to transglutaminase depends on the blockage due to the presence of either other histones or DNA. At low ionic strength, only glutamines on the N-terminal tails of H2B and H3 are labeled with monodansylcadaverine when core particles are incubated with transglutaminase. The partial unfolding that occurs when going to higher ionic strength values results in an increase in the number of reactive glutamines up to a maximum value of 16 per nucleosome. Labeling of some residues (e.g., Gln(104) and Gln(112) of H2A) requires the unwinding of DNA and the dissociation of the H2A--H2B dimers. Gln(76) of H3 is labeled in the H3--H4 tetramer only when the H2A--H2B dimers are dissociated. Interestingly, the labeling of Gln(95) of H2B exclusively depends on the unwinding of DNA. The accurate analysis of these results indicates that the ionic-dependent unwinding of the DNA may occur following a two-state model.

Influence of transglutaminase treatment of skim milk on the formation of ε-(γ-glutamyl)lysine and the susceptibility of individual proteins towards crosslinking

Abstract Results of transglutaminase reaction on the susceptibility of individual milk proteins in skim milk towards crosslinking and on the distinction between crosslinking and other transglutaminase-catalysed reactions, are presented. In unheated milk, transglutaminase had a small effect on proteins whereas in the preheated milk, considerable crosslinking, deamidation and/or amine incorporation occurred. Direct measurement of the dipeptide e -( γ -glutamyl)lysine showed that a rapid crosslinking of proteins occurred during the first 30 min of transglutaminase reaction. Results from the SDS and capillary gel electrophoresis showed that although the crosslinking was prevalent during the entire reaction time, most crosslinking occurred during the first 30 min. In both unheated and preheated milk, major reduction in the monomeric forms of κ - and β -caseins occurred due to the reaction with transglutaminase, suggesting that these two proteins were most susceptible to transglutaminase-induced crosslinking. The high susceptibility of κ -casein towards crosslinking is likely to be at least partly due to its peripheral position in the casein micelles, and the high susceptibility of β -caseins is hypothesised to be due to its dynamic nature and thus ease of accessibility in the micelle structure. In addition, it could be shown that only preheated β -lactoglobulin was susceptible to transglutaminase action, while α -lactalbumin was crosslinked with or without preheating to the same extent.

Properties of deamidated gluten films enzymatically cross-linked.

Films were prepared at neutral pH from deamidated gluten by casting with or without enzymatic treatment by transglutaminase in the presence of various concentrations of diamines added to the film-forming solution. Variation in the glycerol/deamidated gluten ratio from 0.2 to 0.5 had a major effect on the film mechanical properties, which is characteristic of a plasticizing effect. A ratio of 0.35, producing a tensile strength of 1.14 +/- 0.12 MPa and an elongation at break of 376 +/- 62%, was chosen for most of the enzymatic modifications. The action of transglutaminase with or without the addition of external diamines induced a simultaneous increase in tensile strength and elongation at break of the films but tended to decrease the contact angle between the film surface and a water droplet. The presence of diamines in the film solution affected the elongation at break more than the tensile strength of the films. These diamines, able to react at their two extremities, probably acted as spacers between gluten proteins. The decrease in solubility was related to the formation of high molecular weight polymers in the film. The film properties were unaffected by the type of diamine added as secondary substrate in the transglutaminase reaction.

Biochemical Analysis and Rheological Properties of Gluten Modified by Transglutaminase

ABSTRACTA transglutaminase from Streptoverticillium sp. was used to create new covalent intermolecular cross‐links between proteins in gluten. This modification induced drastic changes in its physicochemical properties as well as in its rheological behavior. To understand these changes, we characterized the gluten extractability in acetic acid and identified the proteins of supernatant and pellet by immunoblotting using antibodies specific for each prolamin class. The proportion of soluble proteins decreased drastically after transglutaminase treatment due to the formation of large insoluble polymers as shown by SDS‐PAGE. Among the constitutive proteins of gluten, the high molecular weight glutenin subunits were the most affected in the transglutaminase reaction. The rheological behavior of gluten after 18 hr of incubation with transglutaminase was studied in shear by dynamic measurements over 10‐3 – 101 Hz frequency range and by creep and recovery tests. The behavior of treated glutens remained that of a transient network, but the viscoelastic response was shifted toward shorter times and the steady‐state viscosity was greatly increased. The enzymatic treatment caused a considerable reinforcement of the network. The modified glutens were also less sensitive to thermal processing than unmodified glutens, as shown by a lower amplitude of variation of storage modulus G′ with temperature after enzymatic treatment.

Thymosin β 4 serves as a glutaminyl substrate of transglutaminase. Labeling with fluorescent dansylcadaverine does not abolish interaction with G-actin

Thymosin β 4 possesses actin-sequestering activity and, like transglutaminases, is supposed to be involved in cellular events like angiogenesis, blood coagulation, apoptosis and wound healing. Thymosin β 4 serves as a specific glutaminyl substrate for transglutaminase and can be fluorescently labeled with dansylcadaverine. Two (Gln-23 and Gln-36) of the three glutamine residues were mainly involved in the transglutaminase reaction, while the third glutaminyl residue (Gln-39) was derivatized with a low efficiency. Labeled derivatives were able to inhibit polymerization of G-actin and could be cross-linked to G-actin by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Fluorescently labeled thymosin β 4 may serve as a useful tool for further investigations in cell biology. Thymosin β 4 could provide a specific glutaminyl substrate for transglutaminase in vivo, because of the fast reaction observed in vitro occurring at thymosin β 4 concentrations which are found inside cells. Taking these data together, it is tempting to speculate that thymosin β 4 may serve as a glutaminyl substrate for transglutaminases in vivo and play an important role in transglutaminase-related processes.

Fluorescein Cadaverine Incorporation as a Novel Technique for the Characterization of Terminal Differentiation in Keratinocytes

A novel technique for detecting transglutaminase activity and the production of cornified envelopes in keratinocytes has been devised. This was based on the enzymatic incorporation of fluorescein-labelled cadaverine (FC) into cornified envelopes. The addition of FC (20 μm) to the incubation medium served as an amine donor for transglutaminase reactions in place of protein lysine residues. Cells incorporating the label became visible with fluorescence microscopy and were quantified by fluorimetry. There was a significant difference in the level of FC incorporation into cornified envelopes under the various media conditions and time points employed. The greatest incorporation was observed by keratinocytes cultured in Green's medium and fluorescent intensity decreased in the order: Green's> KGM with calcium>KGM. Confocal imaging of keratinocytes dual stained with FC and propidium iodide revealed the presence of distinct layers and demonstrated how FC was incorporated into differentiating cells and not the basal layer. FC incorporation has the potential to serve as a rapid assessment of terminal differentiation in keratinocytes. It is simple and less time-consuming than currently available alternative techniques. This approach also has the advantage of combining microscopic and quantitative data.

Transglutaminase-Induced Cross-Linking between Subdomain 2 of G-Actin and the 636–642 Lysine-Rich Loop of Myosin Subfragment 1

G-actin was covalently cross-linked with S1 in a bacterial transglutaminase-catalyzed reaction. The cross-linking sites were identified with the help of fluorescent probes and limited proteolysis as the Gln-41 on the DNase I binding loop of subdomain 2 in G-actin and a lysine-rich loop (residues 636–642) on the S1 heavy chain. The same lysine-rich loop was cross-linked to another region of G-actin in a former study (Combeau, C., D. Didry, and M-F. Carlier. 1992. J. Biol. Chem. 267:14038–14046). This indicates the existence of more than one G-actin-S1 complex. In contrast to G-actin, no cross-linking was induced between F-actin and S1 by the transglutaminase reaction. This shows that in F-actin the inner part of the DNase I binding loop, where Gln-41 is located, is not accessible for S1. The cross-linked G-actin-S1 polymerized upon addition of 2 mM MgCl 2 as indicated by electron microscopy and sedimentation experiments. The filaments obtained from the polymerization of cross-linked actin and S1 were much shorter than the control actin filaments. The ATPase activity of the cross-linked S1 was not activated by actin, whereas the K +(EDTA)-activated ATPase activity of S1 was unaffected by the cross-linking. The cross-linking between G-actin and S1 was not influenced by the exchange of the tightly bound calcium to magnesium; however, it was inhibited by the exchange of the actin-bound ATP to ADP. This finding supports the view that the structure of the DNase binding loop in ADP-G-actin is somewhere between the structures of ATP-G-actin and F-actin.

Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretch.

To elucidate the molecular mechanisms whereby expanded polyglutamine stretches elicit a gain of toxic function, we expressed full-length and truncated DRPLA (dentatorubral-pallidoluysian atrophy) cDNAs with or without expanded CAG repeats in COS-7 cells. We found that truncated DRPLA proteins containing an expanded polyglutamine stretch form filamentous peri- and intranuclear aggregates and undergo apoptosis. The apoptotic cell death was partially suppressed by the transglutaminase inhibitors cystamine and monodansyl cadaverine (but not putrescine), suggesting involvement of a transglutaminase reaction and providing a potential basis for the development of therapeutic measures for CAG-repeat expansion diseases.

Polymerization of β-lactoglobulin and bovine serum albumin at oil-water interfaces in emulsions by transglutaminase

Polymerization of β-lactoglobulin and bovine serum albumin at the oil—water interfaces in n-tetradecane-in-water emulsions induced by the transglutaminase reaction was studied. The emulsions were incubated with transglutaminase for various times, and adsorbed and unadsorbed protein fractions at the oil—water interfaces were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. While only monomers were detected in the unadsorbed fractions, polymers were observed in the adsorbed fractions of the both proteins. The sizes and amounts of the polymers increased with incubation time. The incubation with transglutaminase caused much flocculation of the emulsion stabilized by β-lactoglobulin. An increase in viscosity was also observed with the flocculation. The flocculation was probably initiated by the formation of ε-( γ-glutamyl)-lysyl isopeptide bonds between β-lactoglobulin molecules adsorbed on different oil droplets. In the case of the emulsion stabilized by bovine serum albumin, however, the flocculation and the increase in viscosity occurred to only limited extents by the transglutaminase reaction. This suggests that ε-( γ-glutamyl)-lysyl isopeptide bonds induced by the transglutaminase reaction were formed only between neighboring molecules of bovine serum albumin on the same droplet.