Enzymatic ligation for synthesis of single‐chain analogue of monellin by transglutaminase*

Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains: an A chain of 44 amino acid residues and a B chain of 50 residues. Microbial transglutaminase (MTGase) was used for ligation of the monellin subunits without any protecting groups, and without activation of the C^α‐carboxyl group at the C‐terminus. Since a peptide fragment LLQG is a good substrate for MTGase to form an amide bond between the γ‐amide group of the Gln residue and the ε‐amino group of Lys, a monellin B chain analogue in which LLQG was elongated at the C‐terminus (B‐LLQG) was synthesized by solid‐phase synthesis. The monellin A chain analogue in which KGK was elongated at the N‐terminus (KGK‐A) was synthesized by the same method as that of the B chain analogue. The KGK‐A chain and the B‐LLQG chain were coupled by MTGase to give single‐chain analogue of monellin. The single‐chain analogue of monellin was characterized by analytical reverse phase high performance liquid chromatography, electrospray ionization, and amino acid analyses. All analyses gave satisfactory results. The single‐chain analogue of monellin was more heat stable than natural monellin. © 1999 John Wiley & Sons, Inc. Biopoly 50: 193–200, 1999     

A superfamily of archaeal, bacterial, and eukaryotic proteins homologous to animal transglutaminases.

Computer analysis using profiles generated by the PSI-BLAST program identified a superfamily of proteins homologous to eukaryotic transglutaminases. The members of the new protein superfamily are found in all archaea, show a sporadic distribution among bacteria, and were detected also in eukaryotes, such as two yeast species and the nematode Caenorhabditis elegans. Sequence conservation in this superfamily primarily involves three motifs that center around conserved cysteine, histidine, and aspartate residues that form the catalytic triad in the structurally characterized transglutaminase, the human blood clotting factor XIIIa'. On the basis of the experimentally demonstrated activity of the Methanobacterium phage pseudomurein endoisopeptidase, it is proposed that many, if not all, microbial homologs of the transglutaminases are proteases and that the eukaryotic transglutaminases have evolved from an ancestral protease.     

Extracellular matrix modulates mesangial cell apoptosis and mRNA expression of cathepsin-B and tissue transglutaminase

Mesangial matrix is a dynamic structure which modulates mesangial cell function. Since accumulation of matrix precedes the development of focal glomerulosclerosis, we studied the effect of different matrices on mesangial cell (MC) apoptosis. Suspended mesangial cells became apoptotic in a time dependent manner. Collagen type III did not modulate MC apoptosis when compared to cells grown on plastic. MCs grown on Matrigel, collagen type I and IV showed an increased number of apoptotic cells when compared to MCs grown on plastic. DNA end-labeling further confirmed these observations. MCs grown on Matrigel showed enhanced (P < 0.05) mRNA expression for tissue transglutaminase (TTG) and cathepsin-B. Mesangial cells grown on Matrigel also showed enhanced expression of superoxide dismutase (SOD). We conclude that mesangial cells require attachment to the matrix for their survival and alteration of the quality of matrix modulates mesangial cell apoptosis. J. Cell. Biochem. 68:22–30, 1998. © 1998 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.     

Regulation of tissue transglutaminase by prolonged increase of intracellular Ca2+, but not by initial peak of transient Ca2+ increase

Tissue transglutaminase (tTGase) is a member of calcium-dependent transamidation enzyme family, but a detailed regulation mechanism of tTGase by intracellular Ca(2+) is not clearly understood. Arachidonic acid (AA) and maitotoxin (MTX) activated tTGase in a dose- and time-dependent manner. Transfection of tTGase siRNA largely inhibited tTGase expression and tTGase activation by MTX. AA induced an initial increase of intracellular Ca(2+) followed by a prolonged increase. Removal of extracellular Ca(2+) with EGTA blocked the prolonged Ca(2+) increase in response to AA, although the initial Ca(2+) increase remained. In contrast, EGTA completely blocked the increase of intracellular Ca(2+) by MTX. The activation of tTGase by AA or MTX was significantly inhibited by EGTA. Moreover, EGTA prevented the prolonged increase of intracellular Ca(2+) and tTGase activation by lysophosphatidic acid, but had no effect on the initial Ca(2+) increase. These results suggested that tTGase is regulated by the prolonged increase of intracellular Ca(2+) originated from Ca(2+) influx, rather than by the initial peak of transient Ca(2+) increase.     

Transglutaminase 2 in the balance of cell death and survival

Transglutaminase 2 (TG2), a multifunctional enzyme with Ca(2+)-dependent protein crosslinking activity and GTP-dependent G protein functions, is often upregulated in cells undergoing apoptosis. In cultured cells TG2 may exert both pro- and anti-apoptotic effects depending upon the type of cell, the kind of death stimuli, the intracellular localization of the enzyme and the type of its activities switched on. The majority of data support the notion that transamidation by TG2 can both facilitate and inhibit apoptosis, while the GTP-bound form of the enzyme generally protects cells against death. In vivo studies confirm the Janus face of TG2 in the initiation of the apoptotic program. In addition, they reveal a further role: the prevention of inflammation, tissue injury and autoimmunity once the apoptosis has already been initiated. This function of TG2 is partially achieved by being expressed and activated also in macrophages digesting apoptotic cells and mediating a crosstalk between dying and phagocytic cells.     

Isolation,screening, and optimization of bacterial strains for novel transglutaminase production

Transglutaminases are a class of transferases known to form isopeptide bond between glutamine and lysine residues in a protein molecule. Increasing demand for transglutaminase in food and other industries and its low productivity have compelled researchers to isolate and screen micro-organisms with potential to produce it. In the present investigation around 200 isolates were screened for extracellular secretion of microbial transglutaminase (MTGase). Isolate B4 showed enzyme activity of 1.71?±?0.2?U/mL followed by isolate C2 which showed 1.61?±?0.17?U/mL activity, comparable with the activity of industrially used microbial strains. Biochemical analysis along with 16S r-RNA sequencing revealed these isolates (B4 and C2) to be Bacillus nakamurai and a variant of Bacillus subtilis, respectively. Amongst the various production media screened, a medium containing starch and peptone was found best for MTGase production. Correlation between growth, enzyme production, and sugar utilization was also studied and maximum enzyme production was obtained after 48 to 60?hr. Highest MTGase titer (3.95?±?0.03?U/mL for B4 and 2.65?±?0.17?U/mL for C2) was obtained by optimization of parameters. The enzyme was characterized for temperature and pH optima, pH and thermal stability, and effect of metal ions, suggesting its potential use in future applications.     

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall ^1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients ^3,4.In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients ³. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.     

Synthesis of Photolabile Caged Amino Acids for Measuring Amino Acid Transporters

Photolabile precursors (caged compounds) of amino acids such as Ala, Leu, Lys, and Ser were prepared by some simple reactions. These compounds were designed for the rapid, photochemically initiated release of amino acids. These amino acid transporters were expressed in Xenopus oocyte by injecting mRNA prepared from rat kidney. The electrical response of each transporter was examined by applying the amino acids and caged compounds before and after photolysis. Photolysis of the caged amino acids increased the electrical response of the facilitated amino acid transporters expressed in the oocyte. Consequently, these synthesized caged amino acids would be applicable to kinetic investigations on the transporters when combined with a pulsed laser or xenon arc flash lamp.     

Increased Susceptibility to Transglutaminase of Eye Lens Proteins Exposed to Activated Oxygen Species Produced in the Glucose-Glucose Oxidase Reaction

In order to test whether a mild oxidative stress could promote the transglutaminase damaging effect on eye lens proteins, total lens soluble proteins and purified β_L-crystallin have been exposed to H₂O₂: slowly produced by the glucose-glucose oxidase reaction. Soon after the pretreatment, the substrate capacity of the lens proteins for an exogenous transglutaminase has been evaluated. Exposure to the oxidative stress increased the susceptibility of the lens proteins to transglutaminase. When ferrous ions were added to the preincubation medium, in order to convert the H₂O₂ into the hydroxyl radical, the increase was more evident.