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PubMed1977

Human epidermal transglutaminase. II. Immunologic properties.

A monospecific antibody for human epidermal transglutaminase was prepared in rabbits. The antibody formed single immunoprecipitin lines with purified or crude human transglutaminases and quantitatively precipitated transglutaminase activity. There were no precipitin reactions between human factor XIII (zymogen or active enzyme) and antihuman epidermal transglutaminase or between human epidermal transglutaminase and antihuman factor XIII. Heating epidermal transglutaminase (56 degrees C, 15 min) in the presence of calcium increased the enzyme activity up to 10 times baseline levels. The heat-activated human epidermal transglutaminase was identical by immunodiffusion with the native enzyme, although slightly higher precipitation titers were detected following heating. There was no cross-reaction of antihuman epidermal transglutaminase with frog, rat, mouse, chicken, or human hair follicle transglutaminases.

Animals↗

PubMed1978

Endogenous substrates for epidermal transglutaminase.

Potential in vivo substrates for epidermal transglutaminase have been isolated and partially characterized in human stratum corneum and new born rat epidermis. [14C]Putrescine and dansylcadaverine were incorporated into epidermal proteins in vitro. Two high molecular weight proteins incorporated the labels in both the rat ahd human homogenates. One of the proteins was too large to enter a 4% sodium dodecyl sulfate-polyacrylamide spacer gel; the other was seen at the interface between the spacer gel and a 10% sodium dodecyl sulphate-polyacrylamide running gel. These proteins were present in a buffer extract, sodium dodecyl sulphate-dithiothreitol extract and NaOH extract. The labels were also incorporated into protein in the insoluble pellet remaining after the afore-mentioned extractions. The incorporation of putrescine and dansylcadaverine was time dependent, and was inhibited by known inhibitors of epidermal transglutaminase. The two high molecular weight proteins had similar amino acid composition, characterized by high glycine, glutamic acid, serine and aspartic acid. The amino acid composition was similar to, although not identical with, the amino acid composition of alpha-keratin proteins. Epidermal homogenates incubated in the presence of transglutaminase showed progressive insolubilization of the protein. This cross-linking was inhibited by putrescine. [14C]Glycine, [14C]histidine and [4C]proline were incorporated into epidermal proteins in newborn rats in vivo. The glycine-labelled protein became progressively more insoluble when incubated in vitro in the presence of transglutaminase. In vitro incubation with transglutaminase had no effect on the histidine-and proline-labelled proteins.

Amino Acids↗

PubMed1978

Transglutaminase activity in human lymphocytes: early activation by phytomitogens.

Transglutaminase activity is present in human peripheral lymphocytes and is enhanced up to 15-fold within 10-30 min after treatment of the cells with concanavalin A. Phytohemagglutinin has a similar effect. The enzyme is not detected when intact cells are assayed; it is detected only in cell lysates. Concanavalin A enhances transglutaminase activity only when it is incubated with intact cells; concanavalin A treatment of cell lysates has no effect, alpha-Methyl-D-mannoside specifically inhibits the enhancement of transglutaminase activity in cells treated with concanavalin A results from the specific interaction of the lectin with its saccharide binding site on the cell surface, rather than by direct interaction with the enzyme itself. The increased activity of transglutaminase in cells treated with concanavalin A, as compared to unstimulated cells, is maintained under assay conditions in which saturating levels of Ca2+ are present. Transglutaminase may be involved in early cellular events leading to lymphocyte blastogenesis.

Animals↗

PubMed1975

Keratin cross-linking and epidermal transglutaminase. A review with observations on the histochemical and immunochemical localization of the enzyme.

Enzymes known as transglutaminases mediate cross-linking of polypeptide chains by epsilon-(gamma-glutamyl) lysine bonds. Such bonds stabilize structural proteins of many tissues; transglutaminases specific for these tissues have been identified. A calcium- and sulfhydryl-dependent transglutaminase with a molecular weight of 55,000 has been purified from bovine snout epidermis and used to elicit a specific antiserum to the enzyme. Sites of epidermal transglutaminase activity have been localized in the cytoplasm of upper malpighian and granular cells by two complementary methods. When thin-tissue sections were incubated with a fluorescent lysine analog(dansyl cadaverine) and calcium, tissue acceptor sites became fluorescent. Localization was confirmed by fluorescein-conjugated antibody labeling of the enzyme in situ. These observations indicate that epidermal transglutaminase cross-links epidermal proteins during the final stages of keratinization.

Animals↗

PubMed1976

Isolation, purification and characterization of bovine epidermal transglutaminase.

A crosslinking enzyme, epidermal transglutaminase, was isolated from soluble proteins of glabrous cow snout epidermis. This enzyme stabilized fibrin clots rendering them insoluble in 2% acetic acid. It also catalyzed the incorporation of the fluorescent amine, dansyl cadaverine, into casein. Epidermal transglutaminase was purified by chromatography upon DEAE-Sephadex A-50, zone electrophoresis in Pevikon, and Sephadex G-200 gel permeation chromatography. The highly purified substance, which had a specific activity of 3267 amine-incorporating units/mg per h and a molecular weight of 55000, behaved as a single molecular species in the analytical ultracentrifuge. It had a sedimentation coefficient of 4.4 S and migrated as a gamma-globulin at pH 8.6; it displayed anomalous migration in polyacrylamide gels containing sodium dodecyl sulfate. The enzyme was dependent upon free calcium ions and a reduced sulfhydryl group for activity. The apparent Km for dansyl cadaverine was 1.2 - 10(-4) at pH 7.5. Monospecific antiserum to bovine epidermal transglutaminase precipitated with the enzyme in agar. The antiserum prevented fibrin crosslinking but enhanced incorporation of dansyl cadaverine into casein by the enzyme. The epidermal enzyme differed biochemically and immunochemically from bovine plasma transglutaminase (Factor XIII).

Animals↗

PubMed1979

Fibrin membrane endowed with biological function. IV. Formation of cross-links between fibrinogen (or fibrin) and ribonuclease by transglutaminase.

Transglutaminase from guinea pig liver catalyzed the formation of cross-links between fibrinogen (or fibrin) and ribonuclease. Using transglutaminase, immoblized ribonuclease was prepared by two separate methods: (1) fibrinogen-ribonuclease conjugates formed by transglutaminase were treated with thrombin to make fibrin membrane bound covalently to the enzyme; (2) fibrin polymer formed from fibrinogen with thrombin was covalently bound to ribonuclease by transglutaminase to make fibrin-ribonuclease conjugates.

Animals↗

PubMed1977

Rabbit liver transglutaminase: physical, chemical, and catalytic properties.

Transglutaminase (R-glutaminyl-peptide:amine alpha-glutamyl-yltransferase [EC 2.3.2.13]) has been purified to apparent homogeneity from extracts of rabbit liver. The enzyme is a single polypeptide chain of approximately 80 000 molecular weight containing one catalytic site per molecule. That the isolated enzyme is the rabbit counterpart of the well-characterized guinea pig liver transglutaminase is evidenced by the similarities in their amino acid compositions and in their enzymic activities toward several substrates, together with the fact that the isolated rabbit enzyme is immunologically distinct from both rabbit plasma and rabbit platelet blood coagulation factor XIII. A striking difference between the catalytic activities of the rabbit and guinea pig enzymes is the low activity of rabbit transglutaminase for hydroxylamine incorporation into benzyloxycarbonyl-L-glutaminylglycine, a reaction for which the guinea pig enzyme shows a high reactivity. This finding reveals the cause of error in an earlier report (Tyler, H.M., and Laki, K. (1967) Biochemistry 6, 3259) that rabbit liver contains little, if any, of the enzyme. Preparation of, and analytical data on, several glutamine-containing peptide derivatives used in this study are reported here.

Amino Acids↗

PubMed1975

Crosslinking and labeling of membrane proteins by transglutaminase-catalyzed reactions.

Transglutaminase enzymes catalyze for the formation of epsilon(gamma-glutamyl)lysyl crosslinks, or the substitution of a variety of primary amines for the amide function of protein-bound glutaminyl residues. These enzymes should therefore be useful in crosslinking the proteins of membranes and in attaching a variety of chemical probes and labels to these proteins. This usefulness is demonstrated in experiments with the enzyme liver transglutaminase and the membranes with the enzyme liver transglutaminase and the membranes of mouse erythrocytes and of rabbit skeletal muscle sarcoplasmic reticulum.

Animals↗

PubMed1979

Specific fluorescent labeling of chicken myofibril Z-line proteins catalyzed by guinea pig liver transglutaminase.

Guinea pig liver transglutaminase has been found to catalyze the covalent incorporation of dansylcadaverine into chicken skeletal muscle myofibril proteins. Epifluorescence microscopy reveals that the incorporated dansylcadaverine is specifically localized at or near the myofibril Z line. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) indicates that actin constitutes a major fraction of the labeled material; the Z-line proteins alpha-actinin and desmin also show significant labeling, as well as tropomyosin, several additional unidentified proteins, and material with an extremely high molecular weight. The Z-line-specific fluorescence can be removed by brief trypsinization, which releases fluorescent alpha-actinin into the supernate. The majority of the fluorescent protein species are resistant to extraction by either 0.6 M KCl or KI. These results, in conjunction with the microscopic localization, suggest that the dansyl-labeled proteins are constituents of the myofibril Z line. A significant amount of fluorescently labeled transglutaminase is also present in labeled myofibrils, which is resistant to extraction with either 0.6 M KCl or KI. This result indicates a strong, noncovalent interaction between the transglutaminase molecule and the myofibril Z line.

Animals↗

PubMed1979

Differentiation markers in fetal epidermis: transglutaminase and transpeptidase.

Two members of the transpeptidase family of enzymes, transglutaminase and gamma glutamyl transpeptidase, were assayed histochemically and biochemically in developing rat epidermis from day 15 of gestation through postnatal day 5. Electron microscopic examination of serial skin biopsies enabled precise dating of fetal epidermis and periderm and correlation of ultrastructural details of the cells with marker enzyme activities. Transglutaminase activity appeared histochemically in surface epidermis and in hair follicle inner root sheath on day 18 and day 21 of gestation, respectively, concomitant with the onset of terminal keratinization in these tissues. Enzyme activity was biochemically detectable 2 days before the histochemical stain became positive. Transpeptidase was active in fetal epidermis prior to keratinization but was only detectable in basal cells thereafter. Subsequent to birth, enzyme activity rose geometrically in hair follicles undergoing initial differentiation, and was thereafter found in all anagen hairs. Transglutaminase is active only in cells approaching terminal keratinization, while transpeptidase is associated with early phases of epidermal proliferation and differentiation.

Acyltransferases↗

PubMed1975

Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate.

Alcohols↗

PubMed1979

Changes in transglutaminase activity in an experimental model of pulmonary fibrosis induced by paraquat.

An experimental model of pulmonary fibrosis has been developed by dosing rats with one-fifth the LD50 dose of the herbicide paraquat on 5 consecutive days. Approximately 50% of the rats died within 4 days of the completion of dosing, showing macroscopic changes and wet weight increases in the lung consistent with severe oedema. Those animals which died between Days 4 and 10 had markedly increased levels of hydroxyproline in the lung, maximum at Day 6, and increased prolyl hydroxylase activity, maximum at Day 4. These changes, together with an increase in thymidine incorporation into DNA, and increased lung DNA content, were consistent with the development of fibrosis. Measurement of transglutaminase activity in the lung showed marked increases at Days 4 and 10 after completion of dosing. This activity paralleled closely the changes in prolyl hydroxylase activity and became increasingly associated with particulate protein present in the "nuclear pellet" fraction. The presence of zymogen plasma transglutaminase trapped in lung homogenates could not be demonstrated but the contribution by the active plasma transglutaminase (Factor XIIIa) to increases shown at Day 4 cannot be ruled out.

Animals↗

PubMed2026

Noise-Induced Hepatic Stress Is Associated with Transglutaminase Activation and TG7 Upregulation in Rats.

OBJECTIVE: Environmental noise is increasingly recognized as a systemic stressor capable of inducing oxidative and inflammatory responses beyond the auditory system. This study aimed to investigate the effects of chronic noise exposure on transglutaminase (TG) activation, particularly TG7, and its association with hepatic stress responses in rat liver tissue. METHODS: Thirty adult male Wistar albino rats were randomly assigned to control, short-term noise exposure (LT1), and long-term noise exposure (LT2) groups. Gene expression of TG isoforms (TG1, TG2, TG3, TG6, and TG7), inflammatory markers (IL6 and TNF-α), and apoptotic markers (CASP3 and P53) was evaluated using quantitative real-time PCR. Total TG enzymatic activity was assessed colorimetrically. TG7 protein expression and localization were examined by immunohistochemistry and immunofluorescence. DNA integrity was evaluated by agarose gel electrophoresis. Biochemical parameters, including serum malondialdehyde (MDA), interleukin-1 beta (IL1β), cortisone, aspartate aminotransferase, alanine aminotransferase, glucose, insulin, and total cholesterol, were also measured. RESULTS: Noise exposure induced selective upregulation of TG isoforms, with TG7 showing the highest increase (∼10-12-fold). Total transglutaminase enzymatic activity was significantly increased in both noise-exposed groups, with a higher increase in LT1 (*** P < 0.001) and a significant increase in LT2 (** P < 0.01) compared with control, while no significant difference was observed between LT1 and LT2. IL6 and TNF-α increased progressively, particularly in LT2, whereas CASP3 expression was elevated in LT1 but reduced in LT2. DNA analysis revealed mild alterations in genomic integrity without clear internucleosomal fragmentation. TG7 protein showed strong localization within hepatocyte cytoplasm and perisinusoidal regions. Biochemical analysis demonstrated significant increases in MDA (up to 61.28%), IL1β, cortisone, liver enzymes, glucose, insulin, and total cholesterol. CONCLUSION: Chronic noise exposure induces early TG activation, particularly TG7, accompanied by sustained oxidative stress and inflammatory responses in liver tissue. These findings identify TG7 as a potential stress-responsive mediator in noise-induced hepatic injury.

Animals↗

PubMed1976

Role of the intrinsic transglutaminase in the Ca2+-mediated crosslinking of erythrocyte proteins.

Transamidase (i.e., "transglutaminase") activity of human erythrocytes, lysed by a single freezing and thawing to 37 degrees, was measured by a method of incorporating [14C]putrescine into N,N'-dimethylcasein. In the absence of added calcium ions, virtually no enzyme activity could be detected. An increase in concentration of the cation to about 0.5 mM, however, turned on the enzyme to appreciable levels of activity. Simultaneously, Ca2+ produced formation of high molecular weight, nondisulfied bonded protein polymers either directly in the lysate or in fresh cells when the cation was added together with the A23187 ionophore. The polymers could be readily identified in the isolated cell ghosts by means of disc gel electrophoresis. If the Ca2+-promoted formation of polymers was allowed to take place in the presence of 14C-putrescine, then this tracer became incorporated into the polymeric material. The incorporation indicated that polymerization occurred through gamma-glutamyl-epsilon-lysine bridtes. It is suggested that the intrinsic transamidase mediates protein crosslinking of the erythrocyte membrane whenever there is an increase in intracellular Ca2+ concentration. The presence of suitable transglutaminase substrates, e.g. histamine, inhibited crosslinking when the cells were incubated with Ca2+ and ionophore.

Calcium↗

PubMed1978

Relation of protein synthesis and transglutaminase activity to formation of the cross-linked envelope during terminal differentiation of the cultured human epidermal keratinocyte.

When serially cultivated human epidermal keratinocytes are placed in suspension culture they stop growing and form, beneath the plasma membrane, an insoluble envelope consisting of protein cross-linked by epsilon- (gamma-glutamyl)lysine. The formation of envelopes in suspended cells is preceded by a sharp decline in the rate of protein synthesis, and most envelopes appear only after the average rate of protein synthesis has fallen to a very low level. If protein synthesis is reduced over 98 percent with cycloheximide or emetine at the time that surface-grown cells are placed in suspension culture, cross-linked envelopes form in most of the cells. This shows that the precursor of the envelope and the cross-linking enzyme are already in the cytoplasm in most cells of growing surface cultures. The process of envelope formation by suspension cultures is actually accelerated by the inhibitors of protein synthesis; an increased number of cells with cross-linked envelopes is observable within 4-6 h after the addition of cycloheximide. The inhibitor also induces a large fraction of the cells of surface cultures to form enveloped within a few days. These findings suggest that arrest of protein synthesis leads to activation of the cross-linking process. Agents known to inhibit transglutaminase-mediated protein cross-linking-putrescine, iodoacetamide, and ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA)- also prevent envelope formation. Though the activity of the cross-linking transglutaminase depends on the presence of cellular Ca++, we have not been able to activate the cross-linking process by high external Ca++ concentration or ionophores.

Calcium↗

PubMed1978

Transglutaminase-catalyzed cross-linking through diamines and polyamines.

Transglutaminases were found to catalyze the formation of cross-links between peptide chains by means of a transfer reaction between the carboxamide group of a glutamine residue in each chain and both primary amino groups of a diamine or a polyamine. Production of this heretofore undescribed linkage by guinea pig liver transglutaminase was demonstrated by the use of high performance liquid chromatography in a model system using glutamine peptide derivatives and a variety of diamines and polyamines. Evidence for intermolecular cross-linking through polyamines with both the liver enzyme and thrombin-activated human plasma blood coagulation factor XIII was obtained by the use of a guanidinated derivative of beta-casein.

Animals↗

PubMed2026

An efficient endogenous type I-E CRISPR-Cas genome-editing platform for producing transglutaminase in Streptomyces mobaraensis.

Streptomyces mobaraensis is an industrially important actinomycete capable of producing transglutaminase (TGase), a valuable crosslinking enzyme that is widely used in the food, pharmaceutical, and textile industries. However, its genetic manipulation remains challenging owing to the lack of efficient genome-editing tools. Here, we characterized an endogenous type I-E CRISPR-Cas system in S. mobaraensis IPIO2 through bioinformatics analysis and plasmid interference assays, identifying the protospacer adjacent motif as 5'-AAC-3'. We engineered an artificial editing plasmid, pCRISPR, by inserting a mini-CRISPR array (repeat-spacer-repeat) and homologous recombination repair templates into the replicative plasmid pJTU1278. This system exhibited high editing efficiencies, achieving 70% for single-gene deletions and 75-80% for large DNA fragment deletions ranging from 10 to 40 kb. Based on this system, deletion of four genes consistently downregulated during TGase production, identified through comparative proteomics, enhanced TGase production by 8.5-18.5%. Furthermore, deleting the pseudouridimycin and piericidin A1 biosynthetic gene clusters using this system significantly improved the safety profile of TGase production, resulting in a 17% increase in TGase yield. This study established a robust and efficient endogenous CRISPR-Cas-based genome-editing platform in S. mobaraensis, providing a powerful tool for strain engineering and industrial optimization of TGase production.

Comparative proteomics↗

PubMed1979

Specificity of guinea pig liver transglutaminase for amine substrates.

The amine specificity of guinea pig liver transglutaminase, a model enzyme for endo-gamma-glutamine:epsilon-lysin transferases, was explored with the aid of synthetic substrates of high apparent affinities. As exemplified by dansyl- (5-dimethylamino-1-naphthalenesulfonyl), (2,4-dinitrobenzenesulfonyl)-, and (2,4,6-triisopropylbenzenesulfonyl)-cadaverines--each of which showed affinities of approximately 4 x 10(7) M-1--the best amine substrates carried a large hydrophobic substituent attached to an alkylamine side chain of about 7.2 A in length. Altogether, our results point to the importance of a hydrophobic binding region in the enzyme from where the alkyl side chain reaches into a narrow crevice toward the active center and positions the primary amine of the substrate for attacking the carbonyl group of the acyl enzyme intermediate.

Amines↗