Laszlo Lorand

Transglutaminases: crosslinking enzymes with pleiotropic functions

Blood coagulation, skin-barrier formation, hardening of the fertilization envelope, extracellular-matrix assembly and other important biological processes are dependent on the rapid generation of covalent crosslinks between proteins. These reactions — which are catalysed by transglutaminases — endow the resulting supramolecular structure with extra rigidity and resistance against proteolytic degradation. Some transglutaminases function as molecular switches in cytoskeletal scaffolding and modulate protein–protein interactions. Having knowledge of these enzymes is essential for understanding the aetiologies of diverse hereditary diseases of the blood and skin, and various autoimmune, inflammatory and degenerative conditions.

Structural origins of fibrin clot rheology

The origins of clot rheological behavior associated with network morphology and factor XIIIa-induced cross-linking were studied in fibrin clots. Network morphology was manipulated by varying the concentrations of fibrinogen, thrombin, and calcium ion, and cross-linking was controlled by a synthetic, active-center inhibitor of FXIIIa. Quantitative measurements of network features (fiber lengths, fiber diameters, and fiber and branching densities) were made by analyzing computerized three-dimensional models constructed from stereo pairs of scanning electron micrographs. Large fiber diameters and lengths were established only when branching was minimal, and increases in fiber length were generally associated with increases in fiber diameter. Junctions at which three fibers joined were the dominant branchpoint type. Viscoelastic properties of the clots were measured with a rheometer and were correlated with structural features of the networks. At constant fibrinogen but varying thrombin and calcium concentrations, maximal rigidities were established in samples (both cross-linked and noncross-linked) which displayed a balance between large fiber sizes and great branching. Clot rigidity was also enhanced by increasing fiber and branchpoint densities at greater fibrinogen concentrations. Network morphology is only minimally altered by the FXIIIa-catalyzed cross-linking reaction, which seems to augment clot rigidity most likely by the stiffening of existing fibers.

A filter paper assay for transamidating enzymes using radioactive amine substrates

Abstract A filter paper assay was developed for measuring the activity of transamidating enzymes by the incorporation of radioactive amine substrates into proteins. The method is analogous to those generally employed for monitoring the enzymic coupling of isotopic groups to proteins, such as in the ATP-dependent phosphorylation of casein or histone. Use of minimal quantities of reagents, as well as simplicity of handling a large number of test samples, are distinct advantages of this technique. The guinea pig liver transglutaminase-catalyzed incorporation of C14-histamine and C14-putrescine into β-lactoglobulin and casein were studied. The apparent Michaelis kinetics of the reactions permitted evaluation of Km,apps for both isotopic substrates and competitive inhibition constants for nonradioactive amines were also obtained. In contrast to transglutaminase, the fibrinoligase—generated in human plasma from Factor XIII by the addition of thrombin—could not utilize β-lactoglobulin to an appreciable extent. Hence experiments had to be restricted to casein as acceptor protein. Apparent Michaelis constants for the isotopic substrates and inhibition constants for nonradioactive amines could be readily measured also in the complex plasma system. Finally, details of a rapid filter paper assay are described for measuring Factor XIII levels in human plasma by measuring the incorporation of C14-putrescine, at close to saturating concentrations, into casein.

Transglutaminases and Disease: Lessons From Genetically Engineered Mouse Models and Inherited Disorders

The human transglutaminase (TG) family consists of a structural protein, protein 4.2, that lacks catalytic activity, and eight zymogens/enzymes, designated factor XIII-A (FXIII-A) and TG1-7, that catalyze three types of posttranslational modification reactions: transamidation, esterification, and hydrolysis. These reactions are essential for biological processes such as blood coagulation, skin barrier formation, and extracellular matrix assembly but can also contribute to the pathophysiology of various inflammatory, autoimmune, and degenerative conditions. Some members of the TG family, for example, TG2, can participate in biological processes through actions unrelated to transamidase catalytic activity. We present here a comprehensive review of recent insights into the physiology and pathophysiology of TG family members that have come from studies of genetically engineered mouse models and/or inherited disorders. The review focuses on FXIII-A, TG1, TG2, TG5, and protein 4.2, as mice deficient in TG3, TG4, TG6, or TG7 have not yet been reported, nor have mutations in these proteins been linked to human disease.

Factor XIII: structure, activation, and interactions with fibrinogen and fibrin.

Abstract: Fibrin stabilizing factor (factor XIII or FXIII) plays a critical role in the generation of a viable hemostatic plug. Following exposure to thrombin and calcium, the zymogen is activated to FXIIIa that, in turn, catalyzes the formation of Nε(γ‐glutamyl)lysine protein‐to‐protein side chain bridges within the clot network. Introduction of these covalent crosslinks greatly augments the viscoelastic storage modulus of the structure and its resistance to fibrinolytic enzymes. Analysis of the individual reaction steps and regulatory control mechanisms involved in clot stabilization enabled us to reconstruct the entire physiological process. This also serves as a guide for the differential diagnosis of the variety of molecular defects of fibrin stabilization.

Mechanism of allosteric regulation of transglutaminase 2 by GTP

Allosteric regulation is a fundamental mechanism of biological control. Here, we investigated the allosteric mechanism by which GTP inhibits cross-linking activity of transglutaminase 2 (TG2), a multifunctional protein, with postulated roles in receptor signaling, extracellular matrix assembly, and apoptosis. Our findings indicate that at least two components are involved in functionally coupling the allosteric site and active center of TG2, namely (i) GTP binding to mask a conformationally destabilizing switch residue, Arg-579, and to facilitate interdomain interactions that promote adoption of a compact, catalytically inactive conformation and (ii) stabilization of the inactive conformation by an uncommon H bond between a cysteine (Cys-277, an active center residue) and a tyrosine (Tyr-516, a residue located on a loop of the β-barrel 1 domain that harbors the GTP-binding site). Although not essential for GTP-mediated inhibition of cross-linking, this H bond enhances the rate of formation of the inactive conformer.

Dissociation of the subunit structure of fibrin stabilizing factor during activation of the zymogen

Summary Calcium ions play an essential role in the zymogenic conversion of the fibrin stabilizing factor (FSF; Factor XIII of plasma) in that they cause a dissociation of the two types of subunits ( a′ from b ) of the thrombin-activated factor (FSF′). Disc gel electrophoresis carried out under non-denaturating conditions and utilizing a newly developed fluorescent activity staining procedure is eminently suited for demonstrating changes in the quaternary structure of this protein. Also, kinetic evidence is presented to show that the b type of subunit, though enzymatically inactive, plays a role in regulating the rate of the calcium dependent activation of the zymogen.

Transamidating enzymes. II. A continuous fluorescent method suited for automating measurements of factor XIII in plasma

Abstract The covalent incorporation of monodansylcadaverine ( N -5-(aminopentyl)-5-dimethylamino-1-naphthalenesulfonamide) into some proteins, catalyzed by a variety of transamidases, is accompanied by marked changes in the fluorescence emission of the dansyl group. In addition to a blue shift, there is an increase in intensity of fluorescence. Various types of caseins, as well as succinylated β-lactoglobulin or lysozyme, can be used as protein acceptors, though neither of the latter two support the reaction in their native form. The continuous rise of fluorescence intensity made it possible to record directly the course of the enzymic transamidating reactions. As such, the procedure is well suited for automation. Furthermore, the assay is applicable to human plasma for the monitoring of fibrin-stabilizing factor (Factor XIII) levels.

Micromechanical properties of keratin intermediate filament networks

Keratin intermediate filaments (KIFs) form cytoskeletal KIF networks that are essential for the structural integrity of epithelial cells. However, the mechanical properties of the in situ network have not been defined. Particle-tracking microrheology (PTM) was used to obtain the micromechanical properties of the KIF network in alveolar epithelial cells (AECs), independent of other cytoskeletal components, such as microtubules and microfilaments. The storage modulus (G′) at 1 Hz of the KIF network decreases from the perinuclear region (335 dyn/cm2) to the cell periphery (95 dyn/cm2), yielding a mean value of 210 dyn/cm2. These changes in G′ are inversely proportional to the mesh size of the network, which increases ≈10-fold from the perinuclear region (0.02 μm2) to the cell periphery (0.3 μm2). Shear stress (15 dyn/cm2 for 4 h) applied across the surface of AECs induces a more uniform distribution of KIF, with the mesh size of the network ranging from 0.02 μm2 near the nucleus to only 0.04 μm2 at the cell periphery. This amounts to a 40% increase in the mean G′. The storage modulus of the KIF network in the perinuclear region accurately predicts the shear-induced deflection of the cell nucleus to be 0.87 ± 0.03 μm. The high storage modulus of the KIF network, coupled with its solid-like rheological behavior, supports the role of KIF as an intracellular structural scaffold that helps epithelial cells to withstand external mechanical forces.

Cross‐Linking Sites of the Human Tau Protein, Probed by Reactions with Human Transglutaminase

Abstract: A portion of the neurofibrillary tangles of Alzheimers disease has the characteristics of cross‐linked protein. Because the principal component of these lesions is the microtubule‐associated protein tau, and because a major source of cross‐linking activity within neurons is supplied by tissue transglutaminase (TGase), it has been postulated that isopeptide bond formation is a major posttranslational modification leading to the formation of insoluble neurofibrillary tangles. Here we have mapped the sites on two isoforms of human tau protein (τ23 and τ40) capable of participating in human TGase‐mediated isopeptide bond formation. Using dansyl‐labeled fluorescent probes, it was shown that eight Gln residues can function as amine acceptor residues, with two major sites being Gln351 and Gln424. In addition, 10 Lys residues were identified as amine donors, most of which are clustered adjacent to the microtubule‐binding repeats of tau in regions known to be solvent accessible in filamentous tau. The distribution of amine donors correlated closely with that of Arg residues, suggesting a link between neighboring positive charge and the TGase selectivity for donor sites in the protein substrate. Apart from revealing the sites that can be cross‐linked during the TGase‐catalyzed assembly of tau filaments, the results suggest a topography for the tau monomers so assembled.