High molecular weight transglutaminase inhibitor produced by a microorganism (Streptomyces lavendulae Y-200)

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena such as blood clotting, wound healing, apoptosis, and cell differentiation. Streptomyces lavendulae Y-200, isolated from soil, produced a substance that inhibited transglutaminases. The inhibitory substance was purified from the cultured medium by procedures of acid precipitation, deoxyribonuclease treatment, and gel filtration chromatography. The partially purified sample was dark brown. The inhibitory activity was stable under acidic, alkaline, and high temperature conditions, and resistant to the treatment with proteinases such as trypsin and Pronase. The molecular weight of the inhibitory substance was estimated to be between 10(4) and 10(5) from its permeability through ultrafilter membranes. The acid hydrolysate of the inhibitory substance contained amino acids and sugars. The inhibitory substance inhibited both calcium-dependent and calcium-independent transglutaminases in a competitive manner with a glutamine substrate. The extent of inhibition caused by the calcium-dependent transglutaminase increased with increasing calcium concentration. The results obtained here may help identify a novel regulatory substance of transglutaminase in biological systems.     

Molecular Cloning of the Transglutaminase Gene from Bacillus subtilis and Its Expression in Escherichia coli

We cloned and characterized a gene, tgl, encoding transglutaminase in Bacillus subtilis. The tgl gene contained a open reading frame 735-nucleotides long that encoded a 245-residue protein with the molecular weight of 28,300. The deduced amino acid sequence had little sequence similarity with sequences of other transglutaminases from a Streptoverticillium sp. or from mammals. The -10 and -35 regions of a putative promoter resembled the consensus sequence for the σ^K-dependent promoter. In addition, a sequence similar to the consensus sequence for the GerE binding site was found upstream from this region. These findings suggested that tgl was transcribed in the mother cells during a late stage of sporulation. Evidence for this suggestion was that transglutaminase activity was detected in sporulating cells during the same stage. Transglutaminase activity was detected in Escherichia coli cells transformed with a plasmid for expression of the tgl gene.     

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.     

Isolation of Possible Intermediate of Chlorogenic Acid Biosynthesis in Sweet Potato Root Tissue

Thienodolin, a new plant growth-regulating substance, was isolated from the fermentation broth of a streptomycete strain identified as Streptomyces albogriseolus.

The active principle was extracted with ethyl acetate and purified by silica gel column chromatography and preparative HPLC. The substance showed growth promoting activity with 1.2 × 10^?6–1.2 × 10^?5 M treatment to rice seedlings, and inhibitory activity with 4.0 × 10^?5 M treatment.     

Immunoassay of in vitro activated human tissue transglutaminase

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that exerts a variety of physiological functions and is involved in various pathoprocesses. To characterize the role of tTG in disease, simple assays are necessary for detection. We developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) that combines a transamidation step with immunological detection to determine tTG. This assay is based on covalent binding of in vitro activated tTG to N,N′-dimethylated casein and subsequent detection of coupled tTG by specific immunoglobulin G (IgG) antibodies directed against tTG followed by binding of a secondary biotin-labeled antibody. The assay reaches a detection limit of 0.05 ng of tTG/ml. Type 1 and 3 transglutaminases and factor XIII are not detected. By use of this assay, tTG in several cell lines and the stimulatory effect of retinoic acid on tTG expression could be demonstrated. The new assay represents a promising tool for the study of tTG in normal cell physiology and under pathological conditions.     

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.     

Inhibition of transglutaminase by synthetic tyrosine melanin

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. Previously, we found a high molecular mass transglutaminase-inhibitory substance produced by Streptomyces lavendulae Y-200 that appeared to be a melanin substance. Here, we report that synthetic tyrosine melanin inhibited various types of transglutaminases. Tyrosine melanin inhibited tissue-type transglutaminase in a competitive manner with a glutamine substrate, and also inhibited the cross-linking of casein catalyzed by a tissue-type transglutaminase. The melanized hemolymph of the silkworm and melanin solutions prepared from melanin precursors inhibited tissue-type transglutaminase. These results suggested that the melanin substances generally inhibit transglutaminases.     

Targeted Overexpression of Drosophila Transglutaminase-B Induced Characteristic Phenotypes in a Manner Similar to Transglutaminase-A

The Drosophila transglutaminase gene (CG7356) encodes two transglutaminases, dTG-A and dTG-B. To understand the roles of dTG-B during the development of the fly, we examined phenotypes induced through ectopic expression of dTG-B. Overexpression of dTG-B induced rough eye and extra wing crossvein phenotypes. These phenotypes were similar to those observed in the case of targeted overexpression of dTG-A.     

Consequences of terbium (111) binding on the conformation and enzymatic activity of Guinea pig liver transglutaminase

Calcium ions are crucial for expression of transglutaminase activity. Although lanthanides have been reported to substitute for calcium in a variety of protein functions, they did not replace the calcium requirement during transglutaminase activity measurements. Furthermore, lanthanides strongly inhibited purified liver transglutaminase activity using either casein or fibrinogen as substrates. Terbium (III) inhibition of transglutaminase-catalyzed putrescine incorporation into casein was not reversed by the presence of 10–200 fold molar excess of calcium ions (Ki for Tb(III)=60 µM). Conformational changes in purified liver transglutaminase upon Tb(III) binding were evident from a biphasic effect of Tb(III) on transglutaminase binding to fibrin. Low concentrations of Tb(III) (1 µM to 10 µM inhibited the binding of transglutaminase to fibrin, whereas higher concentrations (20 µM to 100 µM promoted binding. Conformational changes in purified liver transglutaminase consequent to Tb(III) binding were also demonstrated by fluorescence spectroscopy due to Forster energy transfer. Fluorescence emission was stable to the presence of 200 mM NaCl and 100 mM CaCl₂ only partially quenched emission. Purified liver transglutaminase strongly bound to Tb(III)-Chelating Sepharose beads and binding could not be disrupted by 100 mM CaCl₂ solution. Our data suggest that Tb(III)-induced conformational changes in transglutaminase are responsible for the observed effects on enzyme structure and function. The potential applications of Tb(III)-transglutaminase interactions in elucidating the structure-function relationships of liver transglutaminase are discussed.     

Transglutaminase and neuronal differentiation

Summary During mouse brain maturation cellular transglutaminase specific activity increases 2.5 fold from day 3 to adulthood. A more pronounced increase is seen during morphological differentiation of mouse neuroblastoma cells, where serum withdrawal induces neurite outgrowth concomitant with a 10 fold increase in transglutaminase specific activity. In contrast, non-dividing neuroblastoma cells lacking neurites show only a 1.5 fold increase in enzyme specific activity. Transglutaminase activity does not reach maximal levels until extensive neurite formation has occurred. More than 80% of the transglutaminase activity is found in the soluble component of brain and neuroblastoma homogenates. Using [³H]-putrescine as the acyl acceptor, endogenous acyl donor substrates in the neuroblastoma cells included proteins that comigrated on SDS-PAGE with tubulin and actin; however, very high molecular weight crosslinked material is the major reaction product in vitro. When purified brain tubulin, microtubule associated proteins and microtubules were compared as exogenous substrates, only the polymeric microtubules were a good acyl donor substrate. Furthermore, preincubation of purified tubulin with transglutaminase and putrescine stimulated both the rate and extent of microtubule assembly. These findings suggest that transglutaminase may mediate covalent cross-linking of microtubules to other cellular components, or the post-translational modification of tubulin by the formation of -glutamylamines.     

Transglutaminase involvement in the secretion of insulin from electropermeabilised rat islets of langerhans

Ca²⁺-Induced insulin release from electropermeabilised islets is inhibited by the transglutaminase inhibitors monodansylcadaverine, glycine methylester, methylamine and cystamine but not by the control compounds dimethyl monodansylcadaverine and sarcosine methylester which lack the primary amine group. Neither monodansylcadaverine nor glycine methylester inhibited insulin secretion induced by either cAMP or the phorbol ester PMA at basal levels (10 nM) of Ca²⁺. These data provide further evidence for the involvement of transglutaminase in Ca²⁺ induced insulin secretion, they also suggest that insulin secretion induced by either cAMP or PMA may act in part by a mechanism independent of that induced by Ca²⁺.     

Biochemical characterization of the medaka (Oryzias latipes) orthologue for mammalian tissue-type transglutaminase (TG2)

Transglutaminase is an enzyme family responsible for post-translational modification such as protein cross-linking and the attachment of primary amine and/or deamidation of glutamine-residue in proteins. Medaka (Oryzias latipes), a recently established model fish, has similar functional proteins to those characterized in mammals. Previously, we found the apparent orthologues that correspond to human transglutaminases in medaka. In this study, regarding the medaka orthologue of human tissue-type transglutaminase (OlTGT), recombinant protein was expressed in an active form in bacteria cultured at low temperature. Using the recombinant protein, we biochemically characterized the enzymatic activity and also obtained a monoclonal antibody that specifically recognized OlTGT. Immunochemical analysis revealed that OlTGT was not expressed ubiquitously, unlike its mammalian orthologue, but in primarily limited tissues such as the eye, brain, spinal cord, and gas gland.     

Purification and Properties of Hydroxycinnamic Acid Ester Hydrolase from Aspergillus japonicus

Hydroxycinnamic acid ester hydrolase from the wheat bran culture medium of Aspergillus japonicus was purified 255-fold by ammonium sulfate fractionation, DEAE-Sephadex treatment and column chromatographies on DEAE-Sephadex, CM-Sephadex and various other Sephadexes. The purified enzyme was free from tannase and found to be homogeneous on polyacrylamide disc gel electrophoresis. Its molecular weight was estimated to be 150,000 by gel filtration and 142,000 by SDS-gel electrophoresis. The isoelectric point of the enzyme was pH 4.80. As to its amino acid composition, aspartic acid and glycine were abundant. The optimum pH and temperature for the enzyme reaction were, respectively, 6.5 and 55°C when chlorogenic acid was used as a substrate. The enzyme was stable between pH 3.0 to 7.5 and inactivated completely by heat treatment at 70°C for 10 min.

All metal ions examined did not activate the enzyme, while Hg⁺⁺ reduced its activity. The enzyme was markedly inhibited by diisopropylfluorophosphate and an oxidizing reagent, iodine, although it was not affected so much by metal chelating or reducing reagents. The purified enzyme hydrolyzed not only esters of hydroxycinnamic acids such as chlorogenic acid, caffeoyl tartaric acid and p-coumaroyl tartaric acid, but also ethyl and benzyl esters of cinnamic acid. However, the enzyme did not act on ethyl esters of crotonic acid and acrylic acid or esters of hydroxybenzoic acids.     

Calcium-dependent affinity purification of transglutaminase from rat liver cytosol

Tissue transglutaminase (E.C.2.3.2.13, R-glutaminyl-peptide: amine glutaminyl transferase), was purified from extracts of rat liver by calcium dependent affinity chromatography on casein-Sepharose. In the presence of 5 mM calcium the enzyme binds to casein Sepharose and is subsequently eluted with 5 mM EGTA. The enzyme has a molecular weight of 83,000 and its activity is dependent on calcium and reduced sulfhydryl residues. A widely distributed calcium-dependent protease (E.C. 3.4.22.17) copurified with transglutaminase by gel filtration and ion exchange chromatography. The separation of these activities prior to chromatography on casein-Sepharose is essential for the isolation of a stable transglutaminase by calcium-dependent affinity chromatography. Affinity chromatography using casein-Sepharose or other immobilized substrates may allow the calcium-dependent purification of a variety of transglutaminases.     

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).     

Metabolic and secretory effects of methylamine in pancreatic islets

The metabolic and secretory effects of methylamine in rat pancreatic islets were investigated. Methylamine accumulated in islet cells, was incorporated into endogenous islet proteins, and inhibited the incorporation of [2,5-³H] histamine into either N,N-dimethylcasein or endogenous islet proteins. Methylamine (2 mM ) did not affect the oxidation of glucose or endogenous nutrients or the intracellular pH in islet cells. Glucose did not affect the activity of transglutaminase in islet homogenates, the uptake of ¹⁴C-methylamine by intact islets or its incorporation into endogenous islet proteins. Methylamine inhibited insulin release evoked by glucose, other nutrient secretagogues, and non-nutrient insulinotropic agents such as L -arginine or gliclazide. The inhibitory effect of methylamine upon insulin release was diminished in the presence of cytochalasin B or at low extracellular pH. Methylamine retarded the conversion of proinsulin to insulin. Trimethylamine (0.7 mM ) was more efficiently taken up by islet cells than methylamine (2.0 mM ), and yet caused only a modest inhibition of insulin release. These findings suggest that methylamine interferes with a late step in the secretory sequence, possibly by inhibiting the access of secretory granules to their exocytotic site.     

Effect of Pepsin Inhibitor (S-PI) on the Growth of Rhodotorula glutinis No. K-24

A tissue-type transglutaminase (TGase) was purified from liver tissue of the red sea bream, Pagrus major, by ion-exchange chromatography and heparin-Sepharose affinity chromatography. Its activity was assessed using a fluorometric assay to measure the incorporation of monodansylcadaverine into N,N′-dimethyl casein. The molecular mass of purified TGase was estimated to be 78kDa by SDS–polyacrylamide gel electrophoresis. The enzyme required Ca²⁺ to express its activity, although 10 mM Sr²⁺ also activated the enzyme fully. TGase activity was maximal at pH 9.0–9.5, and the enzyme was strongly inhibited by sulfhydryl reagents. The purified enzyme catalyzed the cross-linking of myosin heavy chain obtained from Alaska pollack, resulting in gelation of an actomyosin solution. The partial amino acid sequence of this fish TGase showed divisionally significant similarity to TGase from guinea pig liver.     

Role of amphibian egg transglutaminase in the development of secondary cytostatic factor in vitro

Fresh cytosols extracted from unfertilized amphibian eggs contain a cytostatic factor (CSF) which arrests the cell cycle at metaphase when microinjected into cleaving blastomeres. This CSF is sensitive to Ca²⁺, and is designated primary CSF (1°CSF). During storage of Ca²⁺-containing cytosols at 2°C, stable CSF activity appears, designated secondary CSF (2°CSF). In Rana pipiens egg cytosols, the development of 2°CSF coincides with the formation of a protein complex with a molecular weight above 2,000 kDa, and this large molecule exhibits a high 2°CSF activity when purified (Shibuya and Masui, 1989: Development 106:799–808). The present study shows that both the formation of 2°CSF protein complex and the development of its activity are inhibited by ethylamine and glycine-ethyl-ester (GEE), both known as potent transglutaminase (TGase) inhibitors. An affinity-purified polyclonal antibody raised against mammalian transglutaminase reacts with an approximately 68-kDa protein in fresh egg cytosols, as well as with the 2°CSF protein complex. In cytosols deprived of transglutaminase by immunoprecipitation, neither the development of 2°CSF activity nor the formation of its protein complex can occur. These results indicate that transglutaminase of Rana pipiens eggs is responsible for the formation of 2°CSF, and that transglutaminase itself is incorporated into 2°CSF molecules. Mol. Reprod. Dev. 47:302–311, 1997. © 1997 Wiley-Liss, Inc.     

Enzymatic and kinetic properties of blood coagulation factor XIIIa and guinea pig liver transglutaminase utilizing (6-[N-(4-aminobutyl)-N-ethylamino]-2,3-dihydrophthalazine-1,4-dione,as a novel,specific and sensitive chemiluminescent substrate

A novel and sensitive chemiluminescent assay is described to quantitate the acyl transfer activities of blood coagulation factor XIIIa or liver transglutaminase using aminobutyl-N-ethyl-isoluminol as acyl acceptor and N,N′-dimethylcasein, human plasma fibrinogen or fibronectin as acyl donors. The method involved covalently linking aminobutyl-N-ethyl-isoluminol through its free amino group with the γ-carboxamide of protein-bound glutamine resulting in an isopeptide bond; a reaction catalysed by both transglutaminase and factor XIIIa. The protein-bound aminobutyl-N-ethyl-isoluminol was separated from non-conjugated amine by precipitation with trichloroacetic acid. The protein–amine conjugate was dissolved in 500 mmol/L NaOH, oxidized using 15 mmol/L ammonium persulphate and light emission quantitated using a luminometer. Optimal conditions were established to detect factor XIIIa and transglutaminase activities with the chemiluminescent assay. Specificity was demonstrated by lack of activity in the presence of ethylenediamine tetra-acetic acid or unactivated factor XIII, or boiled enzymes, and by competitive inhibition with putrescine and 5′-(biotinamido) pentylamine. The enzymatic and kinetic properties of factor XIIIa and transglutaminase in utilizing aminobutyl-N-ethyl-isoluminol as an acyl acceptor substrate were comprehensively documented. The reaction could be carried out in either a purified system or a complex plasma or cell lysates milieu. The assay is sensitive, specific, and eliminates a need for radioactive reagents. The assay could be used to photolabel reactive glutamines in substrates. The assay could also be adapted to a variety of solid- and solution-phase formats and is amenable to X-ray film and/or light photography imaging. © 1998 John Wiley & Sons, Ltd.     

Purification of endogenous inhibitors of [3H]flunitrazepam binding from bovine brain

Endogenous substances which inhibited the binding of [³H]flunitrazepam ([³H]FNZ) to bovine synaptosomal membranes have been purified from the hot acetic acid extracts of the bovine brain. Three peaks of inhibitory activity were obtained by Sephadex G-10 gel chromatography. Two of the peaks (Peak 2, and Peak 3) which had lower molecular weights that that of peak 1 were identified as inosine and hypoxanthine by TLC methods. Another peak (Peak 1) was further purified to homogeneity using both cation and anion ion-exchange chromatography and the following two-step reversed-phase HPLC. The purified substance inhibited the [³H]FNZ binding dose-dependently and competitively but did not have an effect on the binding of the peripheral-type BZ ligand [³H]Ro 5-4864. It was also shown that the substance was heat-stable and resistant to proteolytic degradation (trypsin, -chymotrypsin, pronase). However, a significant loss of inhibitory activity to [³H]FNZ binding was observed after acid hydrolysis. Molecular weight estimates based on gel filtration methods were less than 500 dalton, and the maximal ultraviolet absorption peak was at 314 nm. These results suggest that this substance is a new endogenous ligand for the central BZ receptor and may play an important role in regulating the GABAergic tone in the central nervous system.