Transglutaminase from UV Mutated Bacillus cereus NRC215: Production, Purification, and Characterization

Transglutaminase (EC 2.3.2.13, TGase) recorded the highest activity (0.101 U/ml) in bacterial isolate NRC215. 16S rRNA sequencing revealed that NRC215 was identified as Bacillus cereus NRC215 under accession number MT229271 in the NCBI database. UV irradiation was employed to improve TGase production. Five rifampin (RIF) resistant mutants were only isolated from UV-treated Bacillus cereus NRC215 for three minutes. The best mutant, BCrif5, exhibiting induced rifampin resistance, gave TGase with higher activity (0.148 U/ml). The ISSR PCR technique was employed to detect these new rearrangements resulting from UV mutagenesis between the wild-type strain and its mutants. Moreover, TGase has been purified by three-step procedures resulting in a recovery of 28 and 34.63% for wild and BCrif5 strains, respectively. The optimal purified TGase activity was exhibited at pH 7 for wild strain while the mutant BCrif5 at pH 5.0 and 40 °C for both wild and BCrif5 strains. Bacillus cereus NRC215 TGase was activated by Ba+2 (102.50 and 107.06%), while it was inhibited by Cu+2 (30% and 22.35%) for wild and BCrif5 strains, respectively. It could be concluded that Bacillus cereus NRC215 is a promising strain for TGase production, which is beneficial as a food additive.

Cytoskeleton, Transglutaminase and Gametophytic Self-Incompatibility in the Malinae (Rosaceae)

Self-incompatibility (SI) is a complex process, one out of several mechanisms that prevent plants from self-fertilizing to maintain and increase the genetic variability. This process leads to the rejection of the male gametophyte and requires the co-participation of numerous molecules. Plants have evolved two distinct SI systems, the sporophytic (SSI) and the gametophytic (GSI) systems. The two SI systems are markedly characterized by different genes and proteins and each single system can also be divided into distinct subgroups; whatever the mechanism, the purpose is the same, i.e., to prevent self-fertilization. In Malinae, a subtribe in the Rosaceae family, i.e., Pyrus communis and Malus domestica, the GSI requires the production of female determinants, known as S-RNases, which penetrate the pollen tube to interact with the male determinants. Beyond this, the penetration of S-RNase into the pollen tube triggers a series of responses involving membrane proteins, such as phospholipases, intracellular variations of cytoplasmic Ca2+, production of reactive oxygen species (ROS) and altered enzymatic activities, such as that of transglutaminase (TGase). TGases are widespread enzymes that catalyze the post-translational conjugation of polyamines (PAs) to different protein targets and/or the cross-linking of substrate proteins leading to the formation of cross-linked products with high molecular mass. When actin and tubulin are the substrates, this destabilizes the cytoskeleton and inhibits the pollen-tube’s growth process. In this review, we will summarize the current knowledge of the relationship between S-RNase penetration, TGase activity and cytoskeleton function during GSI in the Malinae.

Susceptibility of the individual caseins in reconstituted skim milk to cross-linking by transglutaminase: influence of temperature, pH and mineral equilibria

The susceptibility of total casein and the individual caseins in reconstituted skim milk to transglutaminase (TGase)-induced cross-linking was studied as a function of incubation temperature (5–40 °C), pH (5·0–7·0) and mineral addition. Within the ranges studied, the level of total casein cross-linked increased with increasing temperature, pH and concentration of added trisodium citrate, whereas adding calcium chloride had the opposite effect. These effects can be largely related to the effects of these parameters on TGase activity. In addition, the parameters were also found to influence the susceptibility of κ-casein, and to a lesser extent β-casein, to cross-linking, whereas the susceptibility of αs1-casein was not affected. The susceptibility of κ-casein to cross-linking increased with increasing temperature and calcium chloride addition, but decreased with increasing pH and citrate content, whereas the susceptibility of β-casein to TGase-induced cross-linking decreased with increasing temperature, but was not affected by other parameters. These findings highlight the fact that selection of environmental conditions during cross-linking can be applied to tailor the surface, and hence possibly colloidal stability, of casein micelles in TGase-treated milk.

Transglutaminase-1 protects renal epithelial cells from hydrogen peroxide-induced apoptosis through activation of STAT3 and AKT signaling pathways

Our recent studies showed that transglutaminase-1 (TGase-1) is uniquely expressed in mouse renal proximal tubular cells (RPTC) and mediates cell proliferation. In this study, we investigated the role of TGase-1 in cell survival and the survival signaling pathways regulated by TGase-1 in RPTC following oxidant injury. Exposure of RPTC to hydrogen peroxide (H2O2) resulted in apoptosis and an increase in TGase activity. Inhibition of TGase activity with monodansylcadervine (MDC), a TGase inhibitor, or knockdown of TGase-1 with small interference (si)RNA enhanced apoptosis and decreased cell survival in H2O2-treated RPTC. Conversely, overexpression of TGase-1 rendered RPTC more resistant to H2O2 toxicity and MDC treatment blocked this response. Concurrent with RPTC apoptosis, phosphorylation of AKT, signal transducer and activator of transcription-3 (STAT3), and glucogen synthase kinase-3β (GSK-3β) were observed. Pretreatment of cells with MDC or TGase-1 siRNA inhibited phosphorylation of all these molecules. Inhibition of either the AKT or STAT3 pathway potentiated H2O2-induced cell death and increased GSK-3β activity by dephosphorylation at serine 9. Furthermore, treatment with GSK-3β inhibitors reduced H2O2-induced apoptosis and abolished the death-promoting effect of AKT and STAT3 inhibition. Therefore, we have identified TGase-1 as a novel survival factor in renal epithelial cells and it contributes to cell survival through activation of the AKT and STAT3 signaling pathways following oxidant injury.

Kinetin affects the level of chloroplast polyamines and transglutaminase activity during senescence of barley leaves.

We analysed the level of polyamines (PAs) bound to thylakoids and the level and activity of thylakoid transglutaminases throughout barley leaf senescence, retarded by kinetin. The level of PAs bound to thylakoids changed in senescing barley leaves: bound putrescine (PU) and spermidine (SD) increased throughout senescence, whereas bound spermine (SM) decreased. Kinetin diminished the increase in thylakoid-bound PU and SD and almost completely abolished the decrease of the bound SM. These data suggest different roles of PU/SD and SM in thylakoid degradation. Immunodetection of transglutaminases (TGase) in thylakoid fraction revealed three bands of 33, 58 and 78 kDa. During senescence the intensity of all bands increased and it was correlated with an increase in TGase activity. Kinetin down-regulated the accumulation of the 58- and 78-kDa TGases and the TGase activity. We postulate that formation of covalent bonds between PAs and proteins by TGase is involved in chloroplast senescence. The kinetin-mediated preservation of low TGase levels and activity throughout leaf senescence may represent an important component of the mechanism of kinetin action in the retardation of leaf senescence.

Human Tissue Transglutaminase Activity Is Inhibited by Protein Kinase Inhibitors: Potential Therapeutic Implication for Hematological Disorders.

Tissue transglutaminase (TTG) is a multifunctional protein that plays a role in several different hematological processes. TTG is a unique member in transglutaminase gene family in that it exhibits multiple enzymatic properties including transglutaminase (TGase) and GTP/ATP hydrolysis activities. The Ca+2-dependent TGase activity catalyzes an isopeptide bond between a specific γ-glutamyl (Q) containing peptide and ε-amine group from a peptide-bound lysine (K) residue that functions to stabilize proteins and plays a role in wound healing, angiogenesis, cell proliferation, and apoptosis. In the presence of Mg+2, TTG hydrolyzes GTP to GDP, and functions as a G protein (Gαh). TTG can also hydrolyze ATP and functions as a kinase that phosphorylates histones and P53. In contrast to GTP, ATP binding does not inhibit TGase activity and involves distinct binding residues. Although normal activation of TTG is required for normal physiological process, aberrant activation of TGase function is reported to play a role in many inflammatory disorders and has been selected as target for therapeutic intervention to control fibrosis, angiogenesis, inflammation and apoptosis. In an effort to screen for specific small chemical inhibitors of TTG, we investigated a structurally diverse Lopac library (Sigma) containing 1280 pharmacologically active compounds that span a broad range of biological areas. This library contains marketed drugs, failed development candidates and “gold standards” that have well-characterized activities. Initial screening was performed using a solution-phase continuous fluorescent TGase assay performed at 6 μg/ml of purified recombinant human TTG at 37°C for 1 hour in the presence of 10 mM Ca+2 in a 96-well microtiter plate designed for high-throughput screening. In the absence of chemicals, the assay had a 3-fold increase in fluorescent intensity (λEx 340nm; λEm 520nm) when BOC-K-EDA-Dansyl (KXD; a K-substrate) was crosslinked to N, N′-dimethylcasein (a Q substrate). Initially, 20 hits were identified based on inhibition of ≥ 90% of TGase activity at 50 μM of chemicals excluding the well-characterized inhibitors cystamine and iodoacetamide, which target the active site Cys-SH of TTG. These hits were more potent than GTP as 50 μM of GTP only inhibited ≤ 25 % of TGase activity. These hits were subjected to a secondary screening using a colorimetric TGase assay that measured the covalent incorporation of biotinylated pentylamine (BP) into NMC coated microtiter plate. A total of six chemicals designated as LL1 (a c-Raf1 kinase inhibitor), LL2 (a JNK-3 kinase inhibitor), LL3 (a EGFR tyrosine kinase inhibitor), LL4 (protein kinase C and Calmodulin Kinase inhibitors), LL5 (cdc25 phosphatase inhibitor) and LL6 (DNA topoisomerase I inhibitor) were validated. All these inhibitors are known to target kinases and phosphatase by functioning as GTP and ATP analogs with guanine, adenine and quinone as backbone structures. The mechanism of inhibition of TGase activity is under investigation and might be similar to that of GTP. The fact that some kinase and phosphatase inhibitors also inhibit TGase activity further consolidating TTG as a member of kinase supergene family, but also raise a challenging task to develop a specific inhibitor to TTG. However, the chemical inhibitors discovered in current study can be used as a warhead to develop more specific inhibitor of TTG.

Enhanced Expression of Two Novel GTP-Independent Alternatively Spliced Forms of Tissue Transglutaminase in Human Platelets and Leukocytes.

Following vascular injury, blood coagulation and platelet activation trigger the wound healing process. The migration of blood cells through the leaky vessels, formation of new blood vessels, and the synthesis of extracellular matrix (ECM) are essential for tissue repair. Tissue transglutaminase (TTG) is a unique extracellular and intracellular enzyme that stabilizes tissues, binds and releases nitric oxide (NO), and hydrolyzes GTP and ATP. TTG’s crosslinking (TGase) activity makes ECM resistant to protease digestion and aids wound healing. Recent studies demonstrate TTG binds and releases NO inhibiting platelet aggregation and neutrophil migration which inhibiting the inflammatory response. TTG is an enzyme with 687 amino acid residues. The active site involved in protein crosslinking is located at Cys277, while GTP/ATP binding domain is located in the N- and C-terminus and there are 18 free Cys-SH groups distributed throughout the molecule to bind NO. When vascular tissues derived from different phases of wound healing were analyzed on immunoblotting, we detected full-length and truncated forms of TTG antigens. To determine whether the truncated forms of TTG were formed by protein proteolysis or alternative splicing, we screened a human smooth muscle cell lambda cDNA library using a full-length human TTG cDNA as a probe. DNA sequencing analysis of the positive clones revealed that, in additional to wild type, two C-terminal truncated forms, TTG3 and TTG4, were also present. TTG3 and TTG4 were produced by a rare alternate splicing event utilizing alternate 5′ and 3′ splice site, located within exons XII and XIII, respectively. TTG3 and TTG4 were composed of 674 and 646 amino acid residues that shared identical N-terminal 622 amino acids with TTG with distinct 52 and 23 amino acids at the C-terminus that translated into proteins with the predicted Mr of 75 and 70 KDa, respectively. Structure-function studies using purified enzymes demonstrated that TTG3 and TTG4 showed the same calcium requirement as TTG, but had only 9 and 8% of residual TGase activity, respectively. TGase activity of TTG was inhibited by GTP with an IC50 of 6 microM, while both isoforms were not inhibited by up to 400 microM of GTP. GTP also failed to induce a conformational change in the molecule and both isoforms were proteolyzed by tyrpsin while full-length TTG remained intact. Both isoforms retained GTPase and ATPase activities. RT-PCR and immunoblotting demonstrated that TTG3 and TTG4 were expressed at less than 10 and 5% of TTG and were localized in the nucleus in human umbilical vascular endothelial (HUVEC) and vascular smooth muscle (VSMC) cells. In contrast, human leukocytes and platelets contained ~7-fold higher levels of both isoforms than TTG. In conclusion, we identified two novel C-terminal truncated forms of TTG that are expressed by HUVEC, VSMC, human leukocytes and platelets. This is the first report of the expression of two novel TTG isoforms in human blood cells. The different affinity for GTP and TGase activities, distinct intracellular localization and high expression levels in human leukocytes and platelets suggest a unique physiological function of these isoforms during hemostasis. In addition, TTG3 has two additional Cys-SH groups which could bind NO. The physiological significance of both isoforms of TTG in regulating wound repair is currently under investigation.