Transglutaminase Reaction Research Articles

Site-Specific Conjugation of Metal-Chelating Polymers to Anti-Frizzled-2 Antibodies via Microbial Transglutaminase.

Metal-chelating polymer-based radioimmunoconjugates (RICs) are effective agents for radioimmunotherapy but are currently limited by nonspecific binding and off-target organ uptake. Nonspecific binding appears after conjugation of the polymer to the antibody and may be related to random lysine conjugation since the polymers themselves do not bind to cells. To investigate the role of conjugation sites on nonspecific binding of polymer RICs, we developed a microbial transglutaminase reaction to prepare site-specific antibody-polymer conjugates. The reaction was enabled by introducing a Q-tag (i.e., 7M48) into antibody (i.e., Fab) fragments and synthesizing a polyglutamide-based metal-chelating polymer with a PEG amine block to yield substrates. Mass spectrometric analyses confirmed that the microbial transglutaminase conjugation reaction was site-specific. For comparison, random lysine conjugation analogs with an average of one polymer per Fab were prepared by bis-aryl hydrazone conjugation. Conjugates were prepared from an anti-frizzled-2 Fab to target the Wnt pathway, along with a nonbinding specificity control, anti-Luciferase Fab. Fabs were engineered from a trastuzumab-based IgG1 framework and lack lysines in the antigen-binding site. Conjugates were analyzed for thermal conformational stability by differential scanning fluorimetry, which showed that the site-specific conjugate had a similar melting temperature to the parent Fab. Binding assays by biolayer interferometry showed that the site-specific anti-frizzled-2 conjugate maintained high affinity to the antigen, while the random conjugate showed a 10-fold decrease in affinity, which was largely due to changes in association rates. Radioligand cell-binding assays on frizzled-2+ PANC-1 cells and frizzled-2- CHO cells showed that the site-specific anti-frizzled-2 conjugate had ca. 4-fold lower nonspecific binding compared to the random conjugate. Site-specific conjugation appeared to reduce nonspecific binding associated with random conjugation of the polymer in polymer RICs.

Cascade Pumping Overcomes Hydraulic Resistance and Moderates Shear Conditions for Slow Gelatin Fiber Shaping in Narrow Tubes

SummaryIn microextrusion-based 3D bioprinting, shaping gel fibers online, i.e., in narrow tubes, benefits the structural maintenance after extrusion, but it is challenging for materials possessing slow sol-gel transition dynamics. Gelatin, for example, transforms into thermostable fibers via transglutaminase (TG) reaction in as much as 10 min. It causes dramatic flow resistance accumulation and shear stress increase in fluids moving along narrow tubes, resulting in channel clogging and cell detriments. In this study, we overcome the limitations by adopting cascade pumping and performing in a single peristaltic pump that comprises multi-channel pumping units. The pressure and shear stress reduction by over 1-fold are verified by finite element simulation; continuous gelatin fiber production and patterning in a substrate-free manner are achieved via slow online enzymatic cross-linking. The online fiber shaping can be scaled up by numbering up the pumping units and provides another paradigm for biomanufacturing.

Identifying transglutaminase reaction products via mass spectrometry as exemplified by the MUC2 mucin - Pitfalls and traps

In order to demonstrate transglutaminase activity in biological samples immunological as well as glutamine- and amine-donor based assays are commonly used. However, the identification of the transglutaminase reaction product, i. e. the isopeptide cross-linked peptides/proteins or the deamidated protein/peptide are often neglected. This article describes a workflow for the detection of the products of transglutaminase-catalyzed reactions. In particular, possible pitfalls and traps that can arise during the mass spectrometry-based identification of isopeptide cross-links are addressed and characterised on actual samples.

First Step of the Transglutaminase Reaction Catalyzed by Activated Factor XIII Subunit A, Hybrid Quantum Chemistry/Molecular Mechanics Calculations.

The A subunit of blood coagulation factor XIII belongs to the family of transglutaminase enzymes. Its active form (FXIIIa) catalyzes isopeptide bond formation between Glu and Lys residues of specific substrates. Little data are available on the mechanism of this reaction. In this work, the first step of the proposed two-step process was investigated using two different protocols of hybrid QM/molecular mechanics (MM) calculations: an ONIOM-based model as well as QM/MM/molecular dynamics (MD) metadynamics simulations in explicit TIP3P solvent with Gromacs, PLUMED, and a DFTB3 package. Based on calculations involving a truncated system derived from docking of a peptide substrate, our study confirms the higher stability of a zwitterionic form of the catalytic Cys and His residues in the Michaelis complex as well as the "resting" state of the enzyme. Potential energy surfaces, obtained by geometry optimizations with Gaussian, show a two-step reaction mechanism with a zwitterionic tetrahedral intermediate formation in the first and NH3 dissociation in the second step in the case of our ONIOM system. In contrast, in QM/MM MD metadynamics simulations, all three steps occurred in a concerted manner. As a conclusion, our model is able to provide insights into the reaction mechanism of this enzyme.

Design of Lipid-Protein Conjugates Using Amphiphilic Peptide Substrates of Microbial Transglutaminase.

Lipid modification of proteins plays a significant role in regulating the cellular environment. Mimicking natural lipidated proteins is a key technique for assessing the function of proteins modified with lipids and also to render self-assembly of lipids to a target protein. Herein, we report a facile method of conjugating proteins with lipid-fused peptides under homogeneous physiological conditions by using the microbial transglutaminase (MTG) reaction. MTG catalyzes the cross-linking reaction between a specific glutamine (Q) in a protein and a lysine (K) in newly designed lipid-fused peptides. The water-soluble peptide substrates for lipid modification, C14-X-MRHKGS, were newly synthesized, where C14, X, and MRHKGS represent myristic acid, linker peptides composed of G, P, or S, and MTG-reactive K surrounded with basic amino acids, respectively. The MTG-mediated cross-linking reaction between a protein fused with LLQG at the C-terminus and C14-X-MRHKGS (5 molar eq) dissolved in a phosphate saline solution resulted in lipid-protein conjugates with yields of 70 to 100%. The anchoring ability of the obtained lipid-protein conjugates to cell membranes was dependent on the number of G residues in the GnS linker, suggesting that self-assembly and hydrophobicity of the GnS motif serves to enhance membrane anchoring of lipid-protein conjugates.

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications.

Monitoring of transglutaminase crosslinking reaction by 1H NMR spectroscopy on model substrates

Abstract A new method based on 1H NMR spectroscopy was developed for monitoring transglutaminase crosslinking reaction with model molecules (CBZ-Gln-Gly and N-α-acetyl-lysine). The transglutaminase reaction led to the appearance of new resonances on NMR spectrum as well as significant decrease in others. The new observed resonances, originated from newly formed ɛ-(γ-glutamyl)lysine isopeptide bonds, evidence the enzymatic reaction and allow to quantify the ɛ-(γ-glutamyl)lysine fragment. Moreover, the decrease in resonance intensity, originated from lysine, permit to determine the crosslinking degree. These results obtained by 1H NMR spectroscopy can be used as an alternative method to LC–MS, reducing drastically the analysis time from 7 days to 2 h.

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.

Evaluating factor XIII specificity for glutamine-containing substrates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay

Activated factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS)-based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetic assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting only the final deacylation portion of the transglutaminase reaction. With the MALDI–TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, Staphylococcus aureus fibronectin binding protein A, and thrombin-activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P−1 substrate position is sensitive to charge character, and the P−2 and P−3 substrate positions are sensitive to the broad FXIIIa substrate specificity pockets. The more distant P−8 to P−11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase.

Study on the Functional Properties of Soybean Protein Isolate Cross-Linked with Gelatin by Microbial Transglutaminase

A microbial transglutaminase was used to cross-link soybean protein isolates in the presence of gelatin to prepare a cross-linked composite soybean protein-gelatin. Cross-linking conditions, such as total proteins, the ratio of soybean protein isolates to gelatin, and original pH, were fixed at 5% (w/v), 4:1 (w/w), and 7.5, respectively. The 4-hydroxyproline content in the modified product was measured and used as an index to select suitable transglutaminase addition, reaction temperature, and time by single factor experiments, which were found to be 20 U/g proteins, 40°C, and 2 h, respectively. Under the identified conditions, a modified product with 4-hydroxyproline content of about 36 mg/g proteins was prepared, and its functional properties were evaluated. SDS-PAGE analysis showed that several protein polymers existed in the modified product. Compared to the original soybean protein isolates or a cross-linked soybean protein isolates, the modified product had a better emulsifying stability and water holding capacity but a poor enzymatic digestibility in vitro, emulsifying activity and oil binding capacity; meanwhile, the dispersions formed by the modified product exhibited the highest apparent viscosity, storage modulus, and viscous modulus.

Histamine Regulates Binding of Cross-Linked Fibrinogen to Endothelium: Role of Histaminylation in Vascular Biology

Abstract 3304Recent studies have demonstrated that a wide variety of biogenic amines can be covalently cross-linked to intracellular and extracellular proteins. This “aminylation” reaction regulates several important reactions in vascular biology. Serotinylation of small GTPases recently was shown to regulate cell signaling events, platelet activation and promote vascular smooth muscle cell proliferation. The role of extracellular crosslinking of biogenic amines to plasma and extracellular matrix proteins is not well established and could play a role in altering transglutaminase reactions in vascular tissue. Tissue translutaminase (TGM-2) crosslinking of the a-C domain of fibrinogen has been shown to promote clustering of RGD domains, enhance binding of the cross-linked polymers to endothelial cells and promote cell adhesion. While Factor XIIIa prefers to crosslink fibrin, the TGM-2 molecule which is abundantly expressed by erythrocytes, endothelial and vascular smooth muscle cells does not show any preference for fibrin compared to fibrinogen. TGM-2 is a sulfhydryl rich calcium-dependent enzyme that could cross-link a variety of biogenic amines to fibrinogen. We investigated the preference of TGM-2 crosslinking of biogenic amines to fibrinogen in vitro and determined whether the crosslinking would modify the transglutaminase-dependent binding of fibrinogen to endothelial cells. We found histamine was the most effective primary amine inhibitor of the TGM2-mediated cross-linking reaction (Histamine > putrescine >>> serotonin, dopamine, noradrenaline). 1.25 mM histamine inhibited > 75% TGM2-mediated fibrinogen and fibrin cross-linking. The ability of TGM-2 crosslinked fibrinogen complexes to bind to confluent human umbilical vascular endothelial cell (HUVEC) was also studied. Free [125I]-fibrinogen bound to endothelial cells with low affinity, however the binding was increased ∼7 fold when fibrinogen was cross-linked by TGM2. The increase in crosslinked fibrinogen binding was dependent on TGM2 and Ca+2 concentration. Unlabeled crosslinked fibrinogen inhibited the binding by more than 85%. In contrast, unlabeled fibrinogen actually enhanced the binding 1.7 fold suggesting that fibrinogen and cross-linked fibrinogen formed multivalent complexes with cross-linked fibrinogens on the endothelial cell surface. When bound complex were eluted from HUVEC cells after binding experiments, >95% of bound materials were extensively crosslinked and could not enter a 5–15% polyacrylamide gel. In contrast, no high molecular weight material was eluted when non-crosslinked fibrinogen was used in the binding experiments. When fibrinogen was cross-linked in the presence of 8 to 500 micromolar of histamine, the binding was inhibited by ∼75 to 90 %, respectively. In summary, TGM-2 cross-linked fibrinogen showed enhanced binding to endothelial cells as previously reported for purified aC domains of fibrinogen. The binding of fibrinogen to endothelial cells is known to enhance endothelial cell adhesion and leukocyte transmigration, reactions that are involved in wound healing, angiogenesis and inflammation. The TGM-2 crosslinked fibrinogen/fibrinogen complexes may serve as proinflammatory, prothrombotic and proangiogenic factors in vivo. Histaminylation of fibrinogen by TGM-2 could provide a mechanism to regulate these vascular events. Fibrinogen could also serve to control local histamine function as only free histamine can bind to histamine receptors. Transglutaminase mediated histaminylation of fibrinogen could have multiple effects on acute and chronic inflammatory reactions. Disclosures:No relevant conflicts of interest to declare.

Glutamine (Q)‐peptide screening for transglutaminase reaction using mRNA display

Information on subsite specificity of the transglutaminase (TG) is important to design any specific peptides for TG's applications and inhibitor studies. Here, mRNA display was introduced for identifying the subsite specificity of TG from Streptomyces mobaraensis (STG). Functionally active peptides expressed from mRNA display library were differentially conjugated to hexa lysine (K₆-beads according to their relative activities for STG. The active peptide substrates for STG were enriched through six rounds of screening, and its corresponding cDNA/mRNA sequences were identified by DNA sequencing. The results showed that tripeptides such as LQQ and TQP do not show any activity for STG, but the minimum size of the peptide displaying STG activity is pentapeptide. One such predicted peptide sequence, that is, RLQQP (TQ1), showed higher reactivity (ca. 182% conjugation yield) to STG than that of the highly active sequence, that is, control-Q (PQPQLPYPQPQLPY), well-known previously for mammalian TG2. Furthermore, when recombinant DsRed was tagged with TQ1 sequence at its C-terminal, DsRed-TQ1 underwent efficient covalent-immobilization onto alginate-gelatin bead by STG reaction, showing a Q-peptide application as a useful tagging molecule.

Incorporation of 15N-Labeled Ammonia into Glutamine Amide Groups by Protein-Glutaminase and Analysis of the Reactivity for α-Lactalbumin

Protein-glutaminase (PG) is an enzyme that catalyzes the deamidation of protein-bound glutamine residues. We found that an enzyme labeling technique (ELT), which is a stable isotope labeling method based on transglutaminase (TGase) reaction, is applicable for PG. PG catalyzed incorporation of (15)N-labeled ammonium ions into reactive glutamine amide groups in α-lactalbumin similarly to TGase and deamidated the most reactive glutamine amide group once labeled with (15)N. Furthermore, we investigated the effect of ammonium ions on the PG activity by peptide mapping, and more reactive glutamine residues were detected than were detected by the ELT in the presence of ammonium ions. This is probably because ammonium ions are competitive inhibitors, causing decreased reactivity for glutamine residues. We propose the reaction scheme of PG in the presence of the (15)N-labeled ammonium ions and show that the ELT method with PG is useful for evaluating the activity of PG.

Immobilization of alkaline phosphatase on magnetic particles by site-specific and covalent cross-linking catalyzed by microbial transglutaminase

Bacterial alkaline phosphatase (BAP) was site-specifically and covalently immobilized on magnetic particles (MPs) using the enzymatic reaction of microbial transglutaminase (MTG). Immobilization efficiency was affected by the chemical surface treatment of MPs and immobilized BAP exhibited more than 90% of the initial activity after 10 rounds of recycling.

Effects of surface density and size of gold nanoparticles in a fiber-optic localized surface plasmon resonance sensor and its application to peptide detection

In this study, a localized surface plasmon resonance sensor using an optical fiber was fabricated and experimentally proven for the possibility of achieving a biosensor by detecting a combination of peptides using the reaction of transglutaminase. To enhance the performance of a fiber-optic localized surface plasmon resonance (FO LSPR) sensor, we propose to improve the surface condition of an etched optical fiber. A process condition that is able to control the diameter and surface density ratio of gold nanoparticles on the etched optical fiber was established and the sensor with optimized sensitivity was fabricated through optical analysis of varying diameters and surface density ratios of gold nanoparticles. The proposed FO LSPR sensor is expected to measure various biological reactions, and it can be applied to various biological applications.

Impact of transglutaminase reaction on the immunoreactive and sensory quality of yoghurt starter

Cross-linking reaction with transglutaminase (m-TG) was used to obtain yoghurt produced with YO-MIX™ 511 yoghurt starter (Danisco). The use of two different temperatures (37 and 42°C), the presence of m-TG and the storage time (28 days) of the yoghurt were the factors that influenced the immunoreactive and sensory properties. The ELISA results obtained during the reaction of yoghurt proteins with a specific antibody against individual milk proteins (β-lactoglobulin, α-lactalbumin, α-casein, β-casein and κ-casein) indicated differences on the level of immunoreactivity of individual proteins. The β-lg immunoreactivity of yoghurt produced at 37°C was not detectable in any of the samples whereas during the process conducted at 42°C the presence of this protein at low level was observed (1.00 and 3.33% without and with m-TG, respectively). The lack of κ-casein was detected in the storage yogurt sample prepared during fermentation at 42°C. The samples were subjected to sensory evaluation using quantitative descriptive analysis (QDA). A trained personnel (n = 6) rated the yogurts for smell, taste and texture. The data obtained from QDA (16 attributes and 4 yoghurts) were subjected to a principal component analysis (PCA). The results (QDA and PCA) proved that the addition of the m-TG enzyme had significant impact on the sensory quality of the samples. Four principal components (PC1–PC4) determined 96.05% of the total variance of the samples.

Enhanced water absorption of wheat gluten by hydrothermal treatment followed by microbial transglutaminase reaction

The water absorption of wheat gluten plays an important role in the weight, volume and form ratio of the breads. In this paper, hydrothermal treatment and microbial transglutaminase (MTGase) modification were combined to improve the water absorption ratio (WAR) of wheat gluten. To understand the increases in WAR, the changes in MTGase reaction after gluten hydrothermal treatment were also investigated. The sole hydrothermal treatment improved the WAR of gluten. The gluten treated at 100 degrees C for 30 min exhibited the highest WAR value (2.03 g g(-1) gluten) while the WAR of the control without hydrothermal treatment was 1.5 g g(-1) gluten. When gluten was exposed to 90 degrees C for 30 min followed by incubation with MTGase for 5 h, its WAR reached 2.48 g g(-1) gluten. In contrast to control gluten, the surface hydrophobicity of the gluten preheated at 90 degrees C for 30 min increased and fluctuated in a different way during the following MTGase reaction. Meantime, the trend in the amount of soluble protein of preheated gluten was also changed in the progress of MTGase reaction. Hydrothermal treatment followed by MTGase reaction is an efficient approach to improve the WAR of wheat gluten. The analysis of catalytic process, including determination of ammonia, gluten surface hydrophobicity, soluble protein and SDS-PAGE, suggested that hydrothermal pretreatment accelerated the cross-linking reaction and may alter the ratio of gluten deamidation catalysed by MTGase, which induced an increase in the WAR.

“Reverse degradomics”, monitoring of proteolytic trimming by multi‐CE and confocal detection of fluorescent substrates and reaction products

A platform for profiling of multiple proteolytic activities acting on one specific substrate, based on the use of a 96-channel capillary DNA sequencer with CE-LIF of labeled substrate peptides and reaction products is introduced. The approach consists of synthesis of a substrate peptide of interest, fluorescent labeling of the substrate, either aminoterminally by chemical coupling, or carboxyterminally by transglutaminase reaction, proteolysis by a biological mixture of proteases in the absence or presence of protease inhibitors, multi-channel analysis of substrate and reaction products, and data collection and processing. Intact substrate and reaction products, even when varying by only one amino acid, can be relatively semi-quantified in a high-throughput manner, yielding information on proteases acting in complex biological mixtures and without prepurification. Monitoring, classification and inhibition of multiple proteolytic activities are demonstrated on a model substrate, the aminoterminus of the mouse granulocyte chemotactic protein-2. In view of extensive processing of chemokines into various natural forms with different specific biological activities, and of the fragmentary knowledge of processing proteases, examples of processing by neutrophil degranulate, tumor cell culture fluids and plasma are provided. An example of selection and comparison of inhibitory mAbs illustrates that the platform is suitable for inhibitor screening. Whereas classical degradomics technologies analyze the substrate repertoire of one specific protease, here the complementary concept, namely the study of all proteases acting, in a biological context, on one specific substrate, is developed and tuned to identify key proteases and protease inhibitors for the processing of any biological substrate of interest.

Affinity of Microbial Transglutaminase to αs1-, β-, and Acid Casein under Atmospheric and High Pressure Conditions

Kinetics for the reaction of microbial transglutaminase (MTG) with individual caseins in a TRIS-acetate buffer at pH 6.0 was evaluated under atmospheric pressure (0.1 MPa) and high pressure (400 MPa) at 40 °C. The reaction was monitored under the following limitations: The kinetics from the initial velocities was obtained from nonprogressive enzymatic reactions assuming that the individual catalytic constants of reactive glutamine residues are represented by the reaction between MTG and casein monomers. Enzyme reaction kinetics carried out at 0.1 MPa at 40 °C showed Henri-Michaelis-Menten behavior with maximal velocities of 2.7 ± 0.02 × 10(-3), 0.8 ± 0.01 × 10(-3), and 1.3 ± 0.30 × 10(-3) mmol/L · min and K(m) values of 59 ± 2 × 10(-3), 64 ± 3 × 10(-3), and 50 ± 2 × 10(-3) mmol/L for β-, α(s1)-, and acid casein, respectively. Enzyme reaction kinetics of β-casein carried out at 400 MPa and 40 °C also showed a Henri-Michaelis-Menten behavior with a similar maximal velocity of 2.5 ± 0.33 × 10(-3) mmol/L · min, but, comparable to a competitive inhibition, the K(m) value increased to 144 ± 34 × 10(-3) mmol/L. The reaction of MTG with α(s1)-casein under high pressure did not fit in to Henri-Michaelis-Menten kinetics, indicating the complex influence of pressure on protein-enzyme interactions.

Transglutaminases: Widespread Cross-linking Enzymes in Plants

Transglutaminases have been studied in plants since 1987 in investigations aimed at interpreting some of the molecular mechanisms by which polyamines affect growth and differentiation. Transglutaminases are a widely distributed enzyme family catalysing a myriad of biological reactions in animals. In plants, the post-translational modification of proteins by polyamines forming inter- or intra-molecular cross-links has been the main transglutaminase reaction studied. The few plant transglutaminases sequenced so far have little sequence homology with the best-known animal enzymes, except for the catalytic triad; however, they share a possible structural homology. Proofs of their catalytic activity are: (a) their ability to produce glutamyl-polyamine derivatives; (b) their recognition by animal transglutaminase antibodies; and (c) biochemical features such as calcium-dependency, etc. However, many of their fundamental biochemical and physiological properties still remain elusive. It has been detected in algae and in angiosperms in different organs and sub-cellular compartments, chloroplasts being the best-studied organelles. Possible roles concern the structural modification of specific protein substrates. In chloroplasts, transglutaminases appear to stabilize the photosynthetic complexes and Rubisco, being regulated by light and other factors, and possibly exerting a positive effect on photosynthesis and photo-protection. In the cytosol, they modify cytoskeletal proteins. Preliminary reports suggest an involvement in the cell wall construction/organization. Other roles appear to be related to fertilization, abiotic and biotic stresses, senescence and programmed cell death, including the hypersensitive reaction. The widespread occurrence of transglutaminases activity in all organs and cell compartments studied suggests a relevance for their still incompletely defined physiological roles. At present, it is not possible to classify this enzyme family in plants owing to the scarcity of information on genes encoding them.