Legionella effector MavC targets the Ube2N~Ub conjugate for noncanonical ubiquitination.

The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40Ub-Lys92Ube2N) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme's true physiological substrate.

Role of anti-tissue transglutaminase IgA+IgG antibodies in detection of potential celiac disease in patients with type 1 diabetes.

BACKGROUND & OBJECTIVES: : Celiac disease (CD) can exist in various forms in type 1 diabetes (T1D) patients and can remain undetected, leading to severe complications. This study was aimed to evaluate five commercially available anti-tissue transglutaminase (tTG) ELISA kits with distinct formats for the detection of CD and potential CD in T1D patients. Clinical and demographic profiles of the patients with different disease subsets were also studied. METHODS: : Fifty T1D patients with classical and non-classical symptoms of CD and 100 T1D patients without any symptoms of CD were included in this study. Anti-tTG autoantibody levels were estimated by five ELISA kits followed by histological examination of duodenal biopsy. HLA DQ2-DQ8 and DRB1-DQB1 typing was done, and serum levels for transforming growth factor (TGF)-ß1 were also estimated. RESULTS: : Assay format detecting anti-tTG IgA antibodies against recombinant antigens along with neopeptides of gliadin was most efficient in the detection of CD in symptomatic patients, and assay format detecting IgA+IgG helped in the detection of potential CD in asymptomatic T1D patients. These findings were supported by histological examination and human leucocyte antigen analysis. Patients with potential CD were found to have markedly deranged glycaemic control parameters and also had significantly raised serum levels of TGF-ß1, (P <0.05) compared to T1D patients. INTERPRETATION & CONCLUSIONS: : Potential CD can be frequently seen in T1D patients. This can be attributed to the dietary patterns prevalent in the subcontinent and the genetic basis of the disease. Anti-tTG IgA+IgG antibodies can be useful in the detection of these potential CD cases in T1D patients. Early intervention with gluten-free diet can be considered in these patients for better disease management.

In Vivo Measurement of Redox-Regulated TG2 Activity.

Transglutaminase 2 (TG2) is a ubiquitous mammalian enzyme that is implicated in a variety of physiological processes and human diseases. Normally, extracellular TG2 is catalytically dormant due to formation of an allosteric disulphide bond between Cys370 and 371 of the enzyme. In this protocol, we describe a method to reduce this disulphide bond in living mice and to monitor the resulting in vivo TG2 activity. Briefly, exogenous thioredoxin-1 protein (TRX) is prepared and administered as a specific, physiologically relevant reductant of the Cys370-371 disulphide along with the small molecule 5-biotinamidopentylamine (5-BP) as a TG2 activity probe. Tissue cryosections are then analyzed by immunohistochemistry to ascertain the extent of 5-BP incorporation, which serves as a record of the redox state of TG2 in vivo. This protocol focuses on the modulation and measurement of TG2 in the small intestine, but we encourage investigators to evaluate it in their organ(s) of interest.

Separation of two microbial transglutaminase isomers from Streptomyces mobaraensis using pH-mediated cation exchange chromatography and their characterization.

Microbial transglutaminase (MTGase) derived from Streptomyces mobaraensis has been widely used in the food, biotechnology and medicine fields. The lot-to-lot consistency and product stability of MTGase must be ensured. The structure and charge variants of MTGase can influence its bioactivity. In this study, MTGase isomers (MTG I1 and MTG I2) were found during the separation of MTGase by pH-mediated cation-exchange chromatography. MTG I1 and MTG I2 had the same molecular weight and N-terminal amino acid sequences, but they showed charge heterogeneity. The affinity of MTG I2 for substrates was higher than that of MTG I1, and the thermal stability and the acid-base tolerance of MTG I1 were significantly higher than that of MTG I2. Therefore, the ratio of MTG I1/MTG I2 was positively correlated with the stability of MTGase. The buffer pH and the ionic strength of the eluent had significant effects on the separation of MTG I1 and MTG I2, and the elution gradient steepness and column load showed little effect on the separation of the MTG I1 and MTG I2 peaks. We built a stable and repeatable separation method for MTG I1 and MTG I2. MTG I1 could transform into MTG I2, but MTG I2 was unable to transform into MTG I1, making the transformation of MTG I1 to MTG I2 was irreversible. When MTG I2 was removed from the MTGase, a portion of the MTG I1 could transform into MTG I2. Therefore, one way to increase the stability of MTGase was to reduce the transformation of MTG I1 to MTG I2.

Separation of transglutaminase using aqueous two-phase systems composed of two pH-response polymers.

Aqueous two-phase systems (ATPS) have been effectively used as a rapid and economical method for the separation and purification of many enzymes or proteins. However, a key problem is the recovery of the polymers forming ATPS and there are rarely available studies about ATPS for the transglutaminase. In this study, a pH-responsive ATPS has been established by two pH-responsive polymers (PADB4.91 and PADB4.06) that can be recycled by changing the pH values, with high recovery of over 96%. And partitioning of the crude transglutaminase in this new ATPS was investigated for the first time. The main parameters, such as crude TGase feedstock load, the pH of system (pH 6.50-7.80), polymers concentration, and the types and concentration of salts, were studied to optimize partition conditions. In the 3% PADB4.91/2% PADB4.06 ATPS, enzyme recovery of 96.51%, partition coefficient of 4.23 and purification factor of 3.73 for TGase were obtained in the presence of 60 mmol/L MgSO4 and at pH 7.00. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that TGase can be well separated from crude extract.

Purification and biochemical characterization of a novel transglutaminase from Mythimna separata larvae (Noctuidae, Lepidoptera).

A novel transglutaminase (MsTGase) from Mythimna separata larvae was separated and purified; its biochemical property and enzymatic catalytic activities were investigated. MsTGase was obtained chromatographically by the precipitation of Sephadex G-100 gel and DEAE-Cellulose-52 ion-exchange column with 48-fold purification and a reproducible yield of approximately 12%. Molecular weight of the MsTGase was 63.5 KDa and its N-terminal amino acid sequence was GKIEEG-LVI. Michaelis constant of the MsTGase for the substrate N-CBZ-Gln-Gly was 12.83mM with a Vmax of 7.99U/mL. Optimum conditions for MsTGase activity were at 42°C and pH7.5. The enzyme didn't possess metal ion at its catalytic active site; its activity could be significantly inhibited by Mg2+, but activated by Ca2+. Chlorpyrifos and spinosad showed a strong potential to increase MsTGase activity, supporting the view that MsTGase was a novel target. Moreover, the formation of intermolecular cross-links of casein and bovine serum albumin polymerized by MsTGase in the presence of DTT was observed. These findings pave the way for future studies on the physiological role of MsTGase and the potential impact of its regulation on MsTGase-associated pest management.

Molecular interaction mechanisms in reverse micellar extraction of microbial transglutaminase.

Reverse micellar extraction is an efficient and economical alternative for protein purification. In this study, microbial transglutaminase (MTGase) from crude materials was purified using reverse micellar extraction, and the molecular interaction mechanism in reverse micellar extraction of MTGase was explored. By using a molecular simulation study, the interaction mechanism of forward extraction was investigated. The molecular simulation results reveal the interaction of MTGase-water-surfactant is the major driving force for the forward extraction. Further, the effect of ionic strength on molecular interactions in backward extraction was investigated using 1H low-field nuclear magnetic resonance (LF-NMR) and circular dichroism (CD) spectra. In backward extraction, the interactions between water and the other two molecules (MTGase and surfactant molecules) are enhanced while the interactions between target molecules (MTGase) and the other two molecules (water and surfactant molecules) are weakened as the ionic strength increases. Moreover, the effect of size exclusion on backward extraction was also investigated. The results demonstrate size exclusion has limit effect at high ionic strength, and the weakened interaction of MTGase-water-surfactant is the main reason causing the release of the target molecules in backward extraction. This work might provide valuable reference to the MTGase purification and downstream processing.

Transglutaminases Derived from Astrocytes Accelerate Amyloid ß Aggregation.

Activation of astrocytes has been observed in neurodegenerative diseases including Alzheimer's disease (AD). Transglutaminase (TG) is a crosslinking enzyme and contributes to cell adhesion, cytoskeleton construct, extracellular matrix formation, and so on. One of the isozymes, tissue-type TG (TG2) is reported to be activated in AD. Moreover, amyloid ß1-42 (Aß), which is aggregated and the aggregation is detected as characteristic pathology in AD brain, is known to be a substrate of TG2. However, contribution and derivation of TGs in brain for Aß aggregation remain to be clarified. In the present study, we examined the effects of cultured astrocytes prepared from rat embryonic brain cortex on Aß aggregation. When freshly prepared Aß was added to cultured astrocytes for 7 days, Aß monomer decreased and Aß oligomer unchanged. On the other hand, when Aß monomer was diluted with astrocytes conditioned medium, Aß oligomer increased time-dependently, and an inhibitor of TGs, cystamine, blocked it. Furthermore, when cultured astrocytes were stimulated with aggregated Aß, TG2 expression significantly increased. These results suggest that astrocytes could uptake Aß monomer to eliminate from brain; however, TGs derived from astrocytes might accelerate Aß aggregation and the aggregated Aß might enhance TG2 in astrocytes as a vicious cycle in pathological conditions. Adequate control of TGs expression and function in astrocytes would be an important factor in AD pathology.

Transgultaminase-Mediated Nanoarmoring of Enzymes by PEGylation.

PEGylation, the covalent attachment of polyethylene glycol to bioactive molecules, is one of the leading approaches used to prolong pharmacokinetics, to improve the stability, and to reduce the immunogenicity of therapeutic proteins. PEG-conjugated products are associated with better therapy outcomes and improved patient compliance. Widely applied in clinical practice, the technology is mainly used to modify proteins, peptides, and oligonucleotides but also other drug delivery systems such as the liposomal one. Undergoing continuous attempts to optimize therapeutic efficacy and to tune the formation of conjugates, a number of different PEGylation processes are now available to researchers for protein conjugation. Although the possibility of obtaining highly homogeneous conjugate mixtures, preferably formed by a single monoconjugate, from a chemical conjugation reaction continues to be limited, several enzymatic conjugation approaches have recently been investigated to address this need. PEGylation mediated by microbial transglutaminase and its many advantages and modifications are outlined in detail in the current work permitting interested readers to perform site-specific protein derivatization to glutamines or lysines.

Development of a sandwich ELISA assay for quantification of human tissue transglutaminase in cell lysates and tissue homogenates.

Tissue transglutaminase (tTG) belongs to the multigene transglutaminase family of Ca2+-dependent protein cross-linking enzymes. There is a strong evidence that tTG is involved in pathology, such as neurodegenerative diseases, cancer, and celiac disease. To study physiopathological implication of tTG, a sandwich immunoassay has been developed with a new monoclonal antibody for the capture and polyclonal antibody both generated in house. Using this ready to use assay, the tTG protein level can be measured in human tissue homogenates and cells extracts easily in about 4 h. The limit of detection is 1.7 ng/ml; the coefficients of intra- and inter-assay variations range from 1 to 2 % and from 7 to 10 %, respectively. The assay is specific to tTG, and no cross reactivity with TG1, TG3, TG6, TG7, or factor XIIIa was observed. Finally, in the addition to the tTG activity assay previously developed, this assay should be a valuable tool to increase our knowledge of the tTG involvement in physiological and pathological states.

Enzymes from Seafood Processing Waste and Their Applications in Seafood Processing.

Commercial fishery processing results in discards up to 50% of the raw material, consisting of scales, shells, frames, backbones, viscera, head, liver, skin, belly flaps, dark muscle, roe, etc. Besides, fishing operations targeted at popular fish and shellfish species also result in landing of sizeable quantity of by-catch, which are not of commercial value because of their poor consumer appeal. Sensitivity to rapid putrefaction of fishery waste has serious adverse impact on the environment, which needs remedial measures. Secondary processing of the wastes has potential to generate a number of valuable by-products such as proteins, enzymes, carotenoids, fat, and minerals, besides addressing environmental hazards. Fishery wastes constitute good sources of enzymes such as proteases, lipases, chitinase, alkaline phosphatase, transglutaminase, hyaluronidase, acetyl glycosaminidase, among others. These enzymes can have diverse applications in the seafood industry, which encompass isolation and modification of proteins and marine oils, production of bioactive peptides, acceleration of traditional fermentation, peeling and deveining of shellfish, scaling of finfish, removal of membranes from fish roe, extraction of flavors, shelf life extension, texture modification, removal of off-odors, and for quality control either directly or as components of biosensors. Enzymes from fish and shellfish from cold habitats are particularly useful since they can function comparatively at lower temperatures thereby saving energy and protecting the food products. Potentials of these applications are briefly discussed.

Production of soluble and active microbial transglutaminase in Escherichia coli for site-specific antibody drug conjugation.

Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.

High-level expression of a recombinant active microbial transglutaminase in Escherichia coli.

BACKGROUND: Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter. RESULTS: Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ1-8PNP1-20 or LacZ1-8 fusion protein under different promoters. After transformed in competent Escherichia coli K12 strains were fermented in batch and fed-bach mode in different mediums in order to select the best conditions of expression. The two most performing fusion protein systems namely short thermoinducible LacZ1-8Met-MTGase from NP668/1 and long constitutive LacZ1-8PNP1-20Met-MTGase from NP650/1 has been chosen to compare both efficiency of expression and biochemical qualities of the product. Proteins were extracted, purified to homogeneity and verified as a single peak obtained in RP-HPLC. The LacZ1-8PNP1-20Met-MTGase fusion protein purified from NP650/1 exhibited an activity of 15 U/mg compared to 24 U/mg for the shorter fusion protein purified from NP668/1 cell strain. CONCLUSIONS: Combining the experimental data on expression levels and specific activities of purified MTGase fusion proteins, the chimeric LacZ1-8Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.

Discovery of potent and specific dihydroisoxazole inhibitors of human transglutaminase 2.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme that catalyzes the posttranslational modification of glutamine residues on protein or peptide substrates. A growing body of literature has implicated aberrantly regulated activity of TG2 in the pathogenesis of various human inflammatory, fibrotic, and other diseases. Taken together with the fact that TG2 knockout mice are developmentally and reproductively normal, there is growing interest in the potential use of TG2 inhibitors in the treatment of these conditions. Targeted-covalent inhibitors based on the weakly electrophilic 3-bromo-4,5-dihydroisoxazole (DHI) scaffold have been widely used to study TG2 biology and are well tolerated in vivo, but these compounds have only modest potency, and their selectivity toward other transglutaminase homologues is largely unknown. In the present work, we first profiled the selectivity of existing inhibitors against the most pertinent TG isoforms (TG1, TG3, and FXIIIa). Significant cross-reactivity of these small molecules with TG1 was observed. Structure-activity and -selectivity analyses led to the identification of modifications that improved potency and isoform selectivity. Preliminary pharmacokinetic analysis of the most promising analogues was also undertaken. Our new data provides a clear basis for the rational selection of dihydroisoxazole inhibitors as tools for in vivo biological investigation.

Isolation, purification and characterisation of transglutaminase from rosemary (Rosmarinus officinalis L.) leaves.

BACKGROUND: Rosemary (Rosmarinus officinalis L.) is a spice and medicinal herb widely used around the world of the natural antioxidants, and it has been widely accepted as one of the spices with the highest antioxidant activity. Transglutaminase (EC 2.3.2.13: TGase) is an enzyme capable of catalysing acyl transfer reactions by introducing covalent cross-links between proteins, as well as peptides and various primary amines. TGase activity in plants was first observed in pea seedlings, and subsequently found in organs of both lower and higher plants. Recently. TGase has captured researchers' interest due to its attractive potential application in food industries. Therefore, the objectives of this study are isolation and purification of TGase from new plant source rosemary (Rosmarinus officinalis L.) leaves at the laboratory scale. Moreover, investigation of the biochemical properties of the purified TGase to provide a suitable TGase enzyme for food industry applications are in focus. MATERIAL AND METHODS: Rosemary (Rosmarinus officinalis L.) leaves was used as a new plant source to TGase. The biochemical characteristics of the crude and purified enzyme were determined. RESULTS: Rosemary (Rosmarinus officinalis L.) TGase was purified to homogeneity by successive three purification steps including ammonium sulfate precipitatation, ion exchange chromatography on DEAE-Sephadex A-50 column and Size exclusion column chromatography on Sephadex G-100 column. Under experimental conditions. 20-30% of ammonium sulfate saturation in the enzyme solution had a high yield of enzyme activity could be obtained. The purified enzyme from the Sephadex G-100 column had 21.35% yield with increased about 7.31 in purification fold. Rosemary TGase exhibited optimum activity at pH 7.0 and 55°C for the catalytic reaction of hydroxylarnine and Z-Gln-Gly. The purified TGase almost maintained full activity after incubation for 15 ruin up to 60°C and it was completely inactivated at 85°C. The rosemary TGase was stimulated at 2-6 rnM CaCl2 concentrations while it lost about 5-20% from its activity by increasing CaCl2 concentration. Sodium chloride (2-14%) shows no noticeable inhibition of the purified TGase activity. Mg+2, Ba+2 were acivited by the purified TGase while it was str ongly inhibited by Fe+2, moderately by Cir2 and Mn+2. CONCLUSION: This paper reports on the purification and characterisation of TGase from newly isolated plant, rosemary (Rosmarinus officinalis L.) leaves. Finding results of the TGase properties make this enzyme a good candidate for application in the food industry. However, additional work is required to increase activity yield during extraction and purification for commercial scale of TGase from this plant.

Site-specific derivatization of avidin using microbial transglutaminase.

Avidin conjugates have several important applications in biotechnology and medicine. In this work, we investigated the possibility to produce site-specific derivatives of avidin using microbial transglutaminase (TGase). TGase allows the modification of proteins at the level of Gln or Lys residues using as substrate an alkyl-amine or a Gln-mimicking moiety, respectively. The reaction is site-specific, since Gln and Lys derivatization occurs preferentially at residues embedded in flexible regions of protein substrates. An analysis of the X-ray structure of avidin allowed us to predict Gln126 and Lys127 as potential sites of TGase's attack, because these residues are located in the flexible/unfolded C-terminal region of the protein. Surprisingly, incubation of avidin with TGase in the presence of alkylamine containing substrates (dansylcadaverine, 5-hydroxytryptamine) revealed a very low level of derivatization of the Gln126 residue. Analysis of the TGase reaction on synthetic peptide analogues of the C-terminal portion of avidin indicated that the lack of reactivity of Gln126 was likely due to the fact that this residue is proximal to negatively charged carboxylate groups, thus hampering the interaction of the substrate at the negatively charged active site of TGase. On the other hand, incubation of avidin with TGase in the presence of carbobenzoxy-l-glutaminyl-glycine in order to derivatize Lys residue(s) resulted in a clean and high yield production of an avidin derivative, retaining the biotin binding properties and the quaternary structure of the native protein. Proteolytic digestion of the modified protein, followed by mass spectrometry, allowed us to identify Lys127 as the major site of reaction, together with a minor modification of Lys58. By using TGase, avidin was also conjugated via a Lys-Gln isopeptide bond to a protein containing a single reactive Gln residue, namely, Gln126 of granulocyte-macrophage colony-stimulating factor. TGase can thus be exploited for the site-specific derivatization of avidin with small molecules or proteins.

Microbial transglutaminase and its application in the food industry. A review.

The extremely high costs of manufacturing transglutaminase from animal origin (EC 2.3.2.13) have prompted scientists to search for new sources of this enzyme. Interdisciplinary efforts have been aimed at producing enzymes synthesised by microorganisms which may have a wider scope of use. Transglutaminase is an enzyme that catalyses the formation of isopeptide bonds between proteins. Its cross-linking property is widely used in various processes: to manufacture cheese and other dairy products, in meat processing, to produce edible films and to manufacture bakery products. Transglutaminase has considerable potential to improve the firmness, viscosity, elasticity and water-binding capacity of food products. In 1989, microbial transglutaminase was isolated from Streptoverticillium sp. Its characterisation indicated that this isoform could be extremely useful as a biotechnological tool in the food industry. Currently, enzymatic preparations are used in almost all industrial branches because of their wide variety and low costs associated with their biotechnical production processes. This paper presents an overview of the literature addressing the characteristics and applications of transglutaminase.

Comparative characterization of direct and indirect substrate probes for on-chip transamidating activity assay of transglutaminases.

The development of molecular probes is a prerequisite for activity-based protein profiling. This strategy helps in characterizing the catalytic activity and function of proteins, and how these proteins and protein complexes control biological processes of interest. These probes are composed of a reactive functional group and a reporter tag. The reactive group of these substrate probes has been considered to be important to their design, while the significance of the reporter tag is relatively underestimated. In this study we compare TAMRA-cadaverine and biotin-cadaverine, two substrate probes that have different reporter tags but an identical reactive functional group. We assess the on-chip transamidating activity of two transglutaminases; transglutaminase 2 and blood coagulation factor XIII. Activity assays were more easily executed when using the direct probe TAMRA-cadaverine. However the indirect probe, biotin-cadaverine, provided a wider dynamic range, higher signal-to-noise ratio, and lower limit of detection compared to TAMRA-cadaverine. Additionally, we successfully used the on-chip activity assay using the indirect probe to determine TG2 and FXIII activities in Hela cell lysates and human plasma samples, respectively. These results demonstrate that the reporter tag of the substrate probe is critical for protocol execution, sensitivity, and dynamic range of enzyme activity assays. Furthermore, this study provides a helpful guide for development of new probes, which is necessary for the identification of potential biomarkers and therapeutic targets for treating enzyme-related diseases.

Histaminylation of fibrinogen by tissue transglutaminase-2 (TGM-2): potential role in modulating inflammation.

Plasma fibrinogen plays an important role in hemostasis and inflammation. Fibrinogen is converted to fibrin to impede blood loss and serves as the provisional matrix that aids wound healing. Fibrinogen also binds to cytokine activated endothelial cells and promotes the binding and migration of leukocytes into tissues during inflammation. Tissue transglutaminase (TGM-2) released from injured cells could cross-link fibrinogen to form multivalent complexes that could promote adhesion of platelets and vascular cells to endothelium. Histamine released by mast cells is a potent biogenic amine that promotes inflammation. The covalent attachment of histamine to proteins (histaminylation) by TGM-2 could modify local inflammatory reactions. We investigated TGM-2 crosslinking of several biogenic amines (serotonin, histamine, dopamine and noradrenaline) to fibrinogen. We identified histaminylation of fibrinogen by TGM-2 as a preferred reaction in solid and solution phase transglutaminase assays. Histamine caused a concentration-dependent inhibition of fibrinogen cross-linking by TGM-2. Fibrinogen that was not TGM-2 crosslinked bound to unactivated endothelial cells with low affinity. However, the binding was increased by sevenfold when fibrinogen was cross-linked by TGM-2. Histaminylation of fibrinogen also inhibited TGM-2 crosslinking of fibrinogen and the binding to un-activated HUVEC cells by 75­90 %. In summary, the histaminylation of fibrinogen by TGM-2 could play a role in modifying inflammation by sequestering free histamine and by inhibiting TGM-2 crosslinking of fibrinogen.

Two-staged temperature and agitation strategy for the production of transglutaminase from a Streptomyces sp. isolated from Brazilian soils.

Transglutaminase catalyzes the cross-linking reaction between a glutamine residue and a free amine residue of proteins leading to the formation of protein aggregates. In this research, the effects of temperature, agitation, and aeration on the production of transglutaminase in a bench reactor by a newly isolated Streptomyces sp. from Brazilian soils were investigated using a factorial experimental design. The parameters evaluated influenced the enzyme production, and the data showed that the best conditions to enhance cell growth were different from those leading to enhanced transglutaminase production. Thus, a temperature and agitation shift strategy was adopted to increase transglutaminase productivity. The temperature and agitation were first set at 34 °C and 350 rpm, respectively, and after 24 h decreasing to 26 °C and 150 rpm until the end of fermentation. The transglutaminase activity obtained was 2.18 U/mL after 42 h of fermentation, which was twice than that obtained using a constant temperature and agitation fermentation strategy.