The pro-peptide of Streptomyces mobaraensis transglutaminase functions in cis and in trans to mediate efficient secretion of active enzyme from methylotrophic yeasts

Transglutaminase (TGase) from the actinomycete Streptomyces mobaraensis is a useful enzyme in the food industry, and development of an efficient production system for it would be desirable. Herein we report secretion of TGase in an enzymatically active form by methylotrophic yeasts as expression hosts. Secretory production of active TGase required a pro-peptide from TGase. When an artificial Kex2-endopeptidase recognition site was placed between the pro-peptide and mature TGase, secretion and in vitro maturation of TGase depended on Kex2-dependent cleavage. Unexpectedly, coexpression of unlinked pro-peptide with mature TGase yielded efficient secretion of the active enzyme. These results indicate that the pro-peptide from TGase functions not only in an intramolecular but also in an intermolecular manner. Site-directed mutagenesis of putative N-glycosylation sites increased the productivity of the active TGase further. A recombinant Candida boidinii strain was found to secrete active TGase up to 1.83 U/ml (about 90 mg/l) after 119 h of cultivation.     

Thioredoxin motif of Caenorhabditis elegans PDI-3 provides Cys and His catalytic residues for transglutaminase activity

Previous reports have suggested that protein disulfide isomerases (PDIs) have transglutaminase (TGase) activity. The structural basis of this reaction has not been revealed. We demonstrate here that Caenorhabditis elegans PDI-3 can function as a Ca(2+)-dependent TGase in assays based on modification of protein- and peptide-bound glutamine residues. By site-directed mutagenesis the second cysteine residue of the -CysGlyHisCys- motif in the thioredoxin domain of the enzyme protein was found to be the active site of the transamidation reaction and chemical modification of histidine in their motif blocked TGase activity.     

Transglutaminase activity is present in highly purified nonsynaptosomal mouse brain and liver mitochondria

Several active transglutaminase (TGase) isoforms are known to be present in human and rodent tissues, at least three of which, namely, TGase 1, TGase 2 (tissue transglutaminase), and TGase 3, are present in the brain. TGase activity is known to be present in the cytosolic, nuclear, and extracellular compartments of the brain. Here, we show that highly purified mouse brain nonsynaptosomal mitochondria and mouse liver mitochondria and mitoplast fractions derived from these preparations possess TGase activity. Western blotting and experiments with TGase 2 knock-out (KO) mice ruled out the possibility that most of the mitochondrial/mitoplast TGase activity is due to TGase 2, the TGase isoform responsible for the majority of the activity ([14C]putrescine-binding assay) in whole brain and liver homogenates. The identity of the mitochondrial/mitoplast TGase(s) is not yet known. Possibly, the activity may be due to one of the other TGase isoforms or perhaps to a protein that does not belong to the classical TGase family. This activity may play a role in regulation of mitochondrial function both in normal physiology and in disease. Its nature and regulation deserve further study.     

Dissection of Escherichia coli glutamate 5-kinase: functional impact of the deletion of the PUA domain

Glutamate 5-kinase (G5K) catalyzes the controlling first step of the synthesis of the osmoprotective amino acid proline, which feed-back inhibits G5K. Microbial G5K generally consists of one amino acid kinase (AAK) and one PUA (named after pseudo uridine synthases and archaeosine-specific transglycosylases) domain. To investigate the role of the PUA domain, we have deleted it from Escherichia coli G5K. We show that wild-type G5K requires free Mg for activity, it is tetrameric, and it aggregates to higher forms in a proline-dependent way. G5K lacking the PUA domain remains tetrameric, active, and proline-inhibitable, but the Mg requirement and the proline-triggered aggregation are greatly diminished and abolished, respectively, and more proline is needed for inhibition. We propose that the PUA domain modulates the function of the AAK domain, opening the way to potential PUA domain-mediated regulation of G5K; and that this domain moves, exposing new surfaces upon proline binding.     

Structural basis for the coordinated regulation of transglutaminase 3 by guanine nucleotides and calcium/magnesium

Transglutaminase 3 (TGase 3) is a member of a family of Ca2+-dependent enzymes that catalyze covalent cross-linking reactions between proteins or peptides. TGase 3 isoform is widely expressed and is important for effective epithelial barrier formation in the assembly of the cell envelope. Among the nine TGase enzyme isoforms known in the human genome, only TGase 2 is known to bind and hydrolyze GTP to GDP; binding GTP inhibits its transamidation activity but allows it to function in signal transduction. Here we present biochemical and crystallographic evidence for the direct binding of GTP/GDP to the active TGase 3 enzyme, and we show that the TGase 3 enzyme undergoes a GTPase cycle. The crystal structures of active TGase 3 with guanosine 5'-O-(thiotriphosphate) (GTPgammaS) and GDP were determined to 2.1 and 1.9 A resolution, respectively. These studies reveal for the first time the reciprocal actions of Ca2+ and GTP with respect to TGase 3 activity. GTPgammaS binding is coordinated with the replacement of a bound Ca2+ with Mg2+ and conformational rearrangements that together close a central channel to the active site. Hydrolysis of GTP to GDP results in two stable conformations, resembling both the GTP state and the non-nucleotide bound state, the latter of which allows substrate access to the active site.     

Activation of the Ras-ERK pathway inhibits retinoic acid-induced stimulation of tissue transglutaminase expression in NIH3T3 cells

Retinoic acid (RA) is a potent activator of tissue transglutaminase (TGase) expression, and it was recently shown that phosphoinositide 3-kinase (PI3K) activity was required for RA to increase TGase protein levels. To better understand how RA-mediated TGase expression is regulated, we considered whether co-stimulation of NIH3T3 cells with RA and epidermal growth factor (EGF), a known activator of PI3K, would facilitate the induction or increase the levels of TGase expression. Instead of enhancing these parameters, EGF inhibited RA-induced TGase expression. Activation of the Ras-ERK pathway by EGF was sufficient to elicit this effect, since continuous Ras signaling mimicked the actions of EGF and inhibited RA-induced TGase expression, whereas blocking ERK activity in these same cells restored the ability of RA to up-regulate TGase expression. However, TGase activity is not antagonistic to EGF signaling. The mitogenic and anti-apoptotic effects of EGF were not compromised by TGase overexpression, and in fact, exogenous TGase expression promoted basal cell growth and resistance to serum deprivation-induced apoptosis. Moreover, analysis of TGase expression and GTP binding activity in a number of cell lines revealed high basal TGase GTP binding activity in tumor cell lines U87 and MDAMB231, indicating that constitutively active TGase may be a characteristic of certain cancer cells. These findings demonstrate that TGase may serve as a survival factor and RA-induced TGase expression requires the activation of PI3K but is antagonized by the Ras-ERK pathway.     

Cloning and Sequence Analysis of a cDNA Encoding Salmon (Onchorhynchus keta) Liver Transglutaminase

We isolated cDNA clones encoding a transglutaminase (TGase: EC 2.3.2.13) from a salmon (Onchorhynchus keta) cDNA library prepared from the liver. In the cDNA sequence combined, an open reading frame coding for a protein of 680 aa was found. The deduced sequence showed a considerable similarity (62.4%) to that of red sea bream TGase. By comparison of sequence similarity to other TGases, the structure of salmon TGase was like tissue type TGases, rather than membrane-associated type or plasma type TGases. As a structural feature of salmon TGase, 3 aa residues were substituted in the 25 aa sequence around the active site Cys residue, which is conserved among several tissue type TGases. The critical residues thought to form the catalytic-center triad (Cys²⁷², His³³¹, and Asp³⁰¹) were found in the highly conserved region, but the region surrounding Tyr⁵¹¹, which corresponds to the residue participates in hydrogen-bond interactions of active center domain, was less similar to other TGases, except for red sea bream TGase. These findings suggests that the overall structure of fish TGase resembles tissue-type TGases, but has some unique structure.     

Development of a mechanism-based assay for tissue transglutaminase--results of a high-throughput screen and discovery of inhibitors

Tissue transglutaminase (TGase) is a Ca(2+)-dependent enzyme that catalyzes cross-linking of intracellular proteins through a mechanism that involves isopeptide bond formation between Gln and Lys residues. In addition to its transamidation activity, TGase can bind guanosine 5'-triphosphate (GTP) and does so in a manner that is antagonized by calcium. Once bound, GTP undergoes hydrolysis to form guanosine 5'-diphosphate and inorganic phosphate. TGase is thought to play a pathogenic role in neurodegenerative diseases by promoting aggregation of disease-specific proteins that accumulate in these disorders. Thus, this enzyme represents a viable target for drug discovery. We now report the development of a mechanism-based assay for TGase and the results of a screen using this assay in which we tested 56,500 drug-like molecules for their ability to inhibit TGase. In this assay, the Gln- and Lys-donating substrates are N,N-dimethylated casein (NMC) and N-Boc-Lys-NH-CH(2)-CH(2)-NH-dansyl (KXD), respectively. Through a combination of steady state kinetic experiments and reaction progress curve simulations, we were able to calculate values for the initial concentrations of NMC, KXD, and Ca(2+) that would produce a steady state situation in which all thermodynamically significant forms of substrate-bound TGase exist in equal concentration. Under these conditions, the assay is sensitive to both competitive and mixed active-site inhibitors and to inhibitors that bind to the GTP site. The assay was optimized for automated screening in 384-well format and was then used to test our compound library. From among these compounds, 104 authentic hits that represent several mechanistic classes were identified.     

Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptosis in human breast cancer cells

Tissue transglutaminase (TGase) exhibits both a GTP binding/hydrolytic capability and an enzymatic transamidation activity. Increases in TGase expression and activation often occur in response to stimuli that promote cellular differentiation and apoptosis, yet the signaling mechanisms used by these stimuli to regulate TGase expression and activation and the role of TGase in these cellular processes are not well understood. Retinoic acid (RA) consistently induces TGase expression and activation, and it was shown recently that RA-induced TGase expression was inhibited in NIH3T3 mouse fibroblasts co-stimulated with epidermal growth factor (EGF). Here we investigate whether EGF also antagonized RA-induced TGase expression in breast cancer cells. We found that EGF stimulation affected TGase expression and activation very differently in these cancer cells. Not only did EGF fail to block RA-induced TGase expression, but also EGF alone was sufficient to potently up-regulate TGase expression and activation in SKBR3 cells, as well as MDAMB468 and BT-20 cells. Inhibiting phosphoinositide 3-kinase activity severely diminished the ability of EGF and RA to increase TGase protein levels, whereas a constitutively active form of phosphoinositide 3-kinase potentiated the induction of TGase expression by EGF in SKBR3 cells. Because EGF is an established antiapoptotic factor, we examined whether the protection afforded by EGF was dependent on its ability to up-regulate TGase activity in SKBR3 and BT-20 cells. Exposure of cells to a TGase inhibitor or expression of a dominant-negative form of TGase potently inhibited EGF-mediated protection from doxorubicin-induced apoptosis. Moreover, expression of exogenous TGase in SKBR3 cells mimicked the survival advantage of EGF, suggesting that TGase activation is necessary and sufficient for the antiapoptotic properties of EGF. These findings indicate for the first time that EGF can induce TGase expression and activation in human breast cancer cells and that this contributes to their oncogenic potential by promoting chemoresistance.     

Phosphoinositide 3-kinase activity is required for retinoic acid-induced expression and activation of the tissue transglutaminase

Tissue transglutaminase (TGase) is a dual function enzyme that couples an ability to bind GTP with transamidation activity. Retinoic acid (RA) consistently induces TGase expression and activation, and it was recently shown that increased TGase expression protected cells from apoptosis. To better understand how RA regulates TGase, we considered whether RA employed pro-survival signaling pathways to mediate TGase expression and activation. It was found that RA stimulation of NIH3T3 cells activated ERK and phosphoinositide 3-kinase (PI3K); however, only PI3K activation was necessary for RA-induced TGase expression. The overexpression of a constitutively active form of PI3K did not induce TGase expression, indicating that PI3K signaling was necessary but not sufficient for TGase expression. The exposure of cells expressing exogenous TGase to the PI3K inhibitor, LY294002, reduced the ability of TGase to be photoaffinity-labeled with [alpha-(32)P]GTP, providing evidence that PI3K regulates the GTP binding activity of TGase as well as its expression. Moreover, cell viability assays showed that incubation of RA-treated cells with LY294002 together with the TGase inhibitor, monodansylcadaverine (MDC), converted RA from a differentiation factor to an apoptotic stimulus. These findings demonstrate that PI3K activity is required for the RA-stimulated expression and GTP binding activity of TGase, thereby linking the up-regulation of TGase with a well established cell survival factor.     

Overproduction of inactive variants of the murein synthase PBP1B causes lysis in Escherichia coli

Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms (alpha, beta, and gamma) which differ in the length of their N-terminal cytoplasmic region. Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed.     

The pro-region of Streptomyces hygroscopicus transglutaminase affects its secretion by Escherichia coli

Streptomyces transglutaminase (TGase) is secreted as a zymogen (pro-TGase) in liquid cultures and is then processed by the removal of its N-terminal region, resulting in active TGase. To date, there is no report describing TGase (or pro-TGase) secretion in Escherichia coli. In this study, the pro-TGase from Streptomyces hygroscopicus was efficiently secreted by E.?coli BL21(DE3) using the TGase signal peptide or the pelB signal peptide. The secreted pro-TGase was efficiently transformed into active TGase by adding dispase to the culture supernatant of the recombinant strains. Mutational analysis showed that deletion of the first six amino acids of the N-terminal of the pro-region reduced the secretion of pro-TGase, and removal of the next 10 amino acids resulted in the formation of insoluble pro-TGase. These results suggest that the pro-region of TGase is essential for its efficient secretion and solubility in E.?coli.     

Transglutaminase cross-linking properties of the small proline-rich 1 family of cornified cell envelope proteins. Integration with loricrin

Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia. To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no alpha or beta structure but have some organized structure associated with their central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1. The TGase 2 enzyme cross-linked SPR1 proteins poorly. Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo. Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction. Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross-linking by the TGase 1 enzyme. This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models.     

A specific colorimetric assay for measuring transglutaminase 1 and factor XIII activities

Transglutaminase (TGase) is an enzyme that catalyzes both isopeptide cross-linking and incorporation of primary amines into proteins. Eight TGases have been identified in humans, and each of these TGases has a unique tissue distribution and physiological significance. Although several assays for TGase enzymatic activity have been reported, it has been difficult to establish an assay for discriminating each of these different TGase activities. Using a random peptide library, we recently identified the preferred substrate sequences for three major TGases: TGase 1, TGase 2, and factor XIII. In this study, we use these substrates in specific tests for measuring the activities of TGase 1 and factor XIII.     

A prawn transglutaminase: Molecular characterization and biochemical properties

In this study, we report the bioinformatics characterization, gene expression, transglutaminase activity and coagulation assays of transglutaminase (TGase) of freshwater prawn Macrobrachium rosenbergii identified from the constructed cDNA library by GS FLX™ technology. Even though, TGase have sequence similarity, they differ extensively in their substrate specificity and are thought to play an important in variety of functions such as development, tissue differentiation and immune responses etc. Gene expression studies show that MrTGase is widely distributed in the tissues such as heart, muscle, intestine, brain, etc., but higher amounts are found in hemocyte. Results of TGase mRNA relative expression in hemocyte, before and after infected with white spot syndrome baculovirus (WSBV) and Vibrio harveyi show that the gene expression initially increases up to 24 h and then it falls down. Coagulation assay results showed that the endogenous TGase is involved in the rapid assembly of a specific, plasma clotting protein. Structural studies show that MrTGase contains a typical TGc domain between 323 and 424, and two putative integrin-binding motifs at Arg¹⁸⁰–Gly¹⁸¹–Asp¹⁸² and Arg²⁶⁹–Gly²⁷⁰–Asp²⁷¹. The predicted 3D model of MrTGase contains 47.04% coils (366 amino acid residues), 26.74% extended strand (208 residues), 21.72% α-helix (169 residues) and 4.5% beta turns (35 residues). BLASTp analysis of MrTGase exhibited high sequence similarities with other crustacean TGase, with the highest observed in white shrimp (77.1%). Moreover, the phylogenetic analysis also showed that MrTGase clustered with the other members of crustacean TGase. Overall, these results suggested that MrTGase is a major and functional TGase of M. rosenbergii for haemolymph coagulation and also in spread of infection.     

Cover Image,Volume 116, Number 3, March 2019

Transglutaminase (TGase) induces the cross-linking of proteins by catalyzing an acyl transfer reaction. TGase is a zymogen, activated by the removal of its pro-region. Because the pro-region is crucial for folding and inhibition of the TGase activity, the recombinant expression of the mature TGase (mTGase) without the pro-region, usually results in inactive inclusion bodies or low protein yield. Here, Streptomyces netropsis TGase was fused with Escherichia coli lysyl-tRNA synthetase (LysRS), as a module with chaperoning activity in an RNA dependent manner (chaperna). The TGase activity from purified fusion protein induced via the removal of LysRS by tev protease in vitro. Moreover, active mTGase was produced in E. coli via an intracellular cleavage system, wherein LysRS-mTGase was cleaved by the coexpressed tev protease in vivo. The results suggest that LysRS essentially mimics pro-region, which exerts a dual function—folding of TGase into active conformation and keeping it as dormant state—in an RNA-dependent manner. Thus, trans-acting RNAs, prompt the cis-acting chaperone function of LysRS, while being mechanistically similar to the intramolecular chaperone function of the pro-region. These results could be implemented and extended for the folding of “difficult-to-express” recombinant proteins, by harnessing the chaperna function.     

Transglutaminase‐modified wool keratin film and its potential application in tissue engineering

Transglutaminase (TGase) catalyzes the cross‐linking of many proteins and has been widely used to improve the properties of certain protein‐based materials. Keratin is considered as a promising biomaterial candidate following traditional chemical modification. In this study, the effect of TGase on the properties of a wool keratin film was investigated. The TGase‐modified film was applied to drug release and cell proliferation. Treatment with TGase (30 U/g keratin) for 18 h at 40°C increased the tensile strength of the film from 5.18 ± 0.15 MPa to 6.22 ± 0.11 MPa and decreased the elongation at break from 83.47 ± 1.79% to 72.12 ± 3.02%. The stability of the film in PBS and in artificial gastric juice was also improved. A rougher surface and a more compact cross‐section were observed by scanning electron microscopy photographs of the TGase‐treated film. SDS‐PAGE analysis confirmed that higher molecular weight proteins were formed in the TGase‐modified keratin solution and film. The results of the drug release assay using diclofenac indicated that both films with and without TGase treatment led to a high initial release in PBS, which was more constant in artificial gastric juice. The enzyme treatment led to a lower drug release rate from the film. Cell culture experiments suggested that the TGase‐mediated cross‐linked keratin film shows a good biocompatibility and that it can be used for tissue engineering applications.     

Chemistry and biology of the ramoplanin family of peptide antibiotics

The peptide antibiotic ramoplanin factor A2 is a promising clinical candidate for treatment of Gram-positive bacterial infections that are resistant to antibiotics such as glycopeptides, macrolides, and penicillins. Since its discovery in 1984, no clinical or laboratory-generated resistance to this antibiotic has been reported. The mechanism of action of ramoplanin involves sequestration of peptidoglycan biosynthesis Lipid intermediates, thus physically occluding these substrates from proper utilization by the late-stage peptidoglycan biosynthesis enzymes MurG and the transglycosylases (TGases). Ramoplanin is structurally related to two cell wall active lipodepsipeptide antibiotics, janiemycin, and enduracidin, and is functionally related to members of the lantibiotic class of antimicrobial peptides (mersacidin, actagardine, nisin, and epidermin) and glycopeptide antibiotics (vancomycin and teicoplanin). Peptidomimetic chemotherapeutics derived from the ramoplanin sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium (VRE), methicillin-resistant Staphylococcus aureus (MRSA), and related pathogens. Here we review the chemistry and biology of the ramoplanins including its discovery, structure elucidation, biosynthesis, antimicrobial activity, mechanism of action, and total synthesis.     

Lytic transglycosylases: bacterial space-making autolysins

Lytic transglycosylases are an important class of bacterial enzymes that act on peptidoglycan with the same substrate specificity as lysozyme. Unlike the latter enzymes, however, the lytic transglycosylases are not hydrolases but instead cleave the glycosidic linkage between N-actetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anydromuramoyl product. They are ubiquitous in bacteria which produce a compliment of different forms that are responsible for creating space within the peptidoglycan sacculus for its biosynthesis and recycling, cell division, and the insertion of cell-envelope spanning structures, such as flagella and secretion systems. As well, the lytic transglyosylases may have a role in pathogenesis of some bacterial species. Given their important role in bacterial cell wall metabolism, the lytic transglycosylases may present an attractive new target for the development of broad-spectrum antibiotics.