Secondary literature sources for the TGc domain

For each of the hand selected primary papers associated with the TGc domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

Human procarboxypeptidase U, or thrombin-activable fibrinolysis inhibitor, is a substrate for transglutaminases. Evidence for transglutaminase-catalyzed cross-linking to fibrin.

Valnickova Z, Enghild JJ
J Biol Chem. 1998; 273: 27220-4

Procarboxypeptidase U (EC 3.4.17.20) (pro-CpU), also known as plasma procarboxypeptidase B and thrombin-activable fibrinolysis inhibitor, is a human plasma protein that has been implicated in the regulation of fibrinolysis. In this study, we show that pro-CpU serves as a substrate for transglutaminases. Both factor XIIIa and tissue transglutaminase catalyzed the polymerization of pro-CpU and the cross-linking to fibrin as well as the incorporation of 5-dimethylaminonaphthalene-1-sulfonyl cadaverine (dansylcadaverine), [14C]putrescine, and dansyl-PGGQQIV. These findings show that pro-CpU contains both amine acceptor (Gln) and amine donor (Lys) residues. The amine acceptor residues were identified as Gln2, Gln5, and Gln292, suggesting that both the activation peptide and the mature enzyme participate in the cross-linking reaction. These observations imply that transglutaminases may mediate covalent binding of pro-CpU to other proteins and cell surfaces in vivo. In particular, factor XIIIa may cross-link pro-CpU to fibrin during the latter part of the coagulation cascade, thereby helping protect the newly formed fibrin clot from premature plasmin degradation. Moreover, the cross-linking may facilitate the activation of pro-CpU, stabilize the enzymatic activity, and protect the active enzyme from further degradation.

A simple and rapid screen for inhibitors of factor XIIIa.

Tymiak AA, Tuttle JG, Kimball SD, Wang T, Lee VG
J Antibiot (Tokyo). 1993; 46: 204-6

Transamidase kinetics. Amide formation in the enzymic reactions of thiol esters with amines.

Stenberg P, Curtis CG, Wing D, Tong YS, Credo RB, Gray A, Lorand L
Biochem J. 1975; 147: 153-63

1. Beta-Phenylpropionylthiocholine and N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylcadaverine) serve as a pair of water-soluble (pH7.5) model substrates for transamidating enzymes. Amide formation could be followed directly through fluorescence measurements by monitoring the continuous extraction of the water-soluble coupling product, N-(beta-phenylpropionyl)dansylcadaverine, into n-heptane. By this procedure, the steady-state kinetics of glutamine-lysine endo-gamma-glutamyltransferase from human plasma (fibrinoligase, thrombin- and Ca2+-activated blood coagulation Factor XII) and from guinea-pig liver (liver transglutaminase) were investigated at 25 degrees C. 2. With beta-phenylpropionylthiocholine as the varied substrate, Lineweaver-Burk plots with various concentrations of dansylcadaverine intercept on the horizontal axis, suggesting that formation of the acyl-enzyme is rate limiting. 3. On the basis of functional normality of active sites, kcat. values of 1.8 s(-1) and 0.9 s(-1) were obtained for the plasma and liver gamma-glutamyltransferase respectively. The two enzymes show identical affinities for the first substrate, beta-phenylpropionylthiocholine, with Ka 4 times 10(-4) M. 4. Utilization of the second substrate, dansylcadaverine, appears to be an order of magnitude more efficient with the liver enzyme. 5. N-(5-Amino-3-thiapentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylthiacadaverine) could be used instead of dansylcadaverine in the fluorescent kinetic system. 6. Competitive inhibition by a non-fluorescent amine substrate histamine was also evaluated.

Plant transglutaminases.

Serafini-Fracassini D, Del Duca S, Beninati S
Phytochemistry. 1995; 40: 355-65

The identification procedures, the characteristics and the potential function of the recently detected plant transglutaminases, are discussed in the light of the knowledge of animal transglutaminases. The enzyme has been studied occasionally in lower organisms (bacteria, fungi and green algae) and more extensively in Angiosperms.

Vitronectin is a substrate for transglutaminases.

Sane DC, Moser TL, Pippen AM, Parker CJ, Achyuthan KE, Greenberg CS
Biochem Biophys Res Commun. 1988; 157: 115-20

Vitronectin (VN) was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (TG). Incorporation of [3H]-putrescine indicated the presence of reactive glutaminyl residues in VN. When VN was incubated with TG or Factor XIIIa, in the absence of putrescine, multimeric covalent complexes were identified, indicating that VN can also contribute lysyl residues to the bond catalyzed by transglutaminases. Cross-linking of VN by TG and Factor XIIIa may modulate the effects of VN on the complement and coagulation systems in hemostatic plugs and extracellular matrix.

Primary structure of keratinocyte transglutaminase.

Phillips MA, Stewart BE, Qin Q, Chakravarty R, Floyd EE, Jetten AM, Rice RH
Proc Natl Acad Sci U S A. 1990; 87: 9333-7

The nucleotide and deduced amino acid sequences of the coding regions of human and rat keratinocyte transglutaminases (protein-glutamine: amine gamma-glutamyltransferase; EC 2.3.2.13) have been determined. These yield proteins of approximately 90 kDa that are 92% identical, indicative of the conservation of important structural features. Alignments of amino acid sequences show substantial similarity among the keratinocyte transglutaminase, human clotting factor XIII catalytic subunit, guinea pig liver tissue transglutaminase, and the human erythrocyte band-4.2 protein. The keratinocyte enzyme is most similar to factor XIII, whereas the band-4.2 protein is most similar to the tissue transglutaminase. A salient feature of the keratinocyte transglutaminase is its 105-residue extension beyond the N terminus of the tissue transglutaminase. This extension and the unrelated activation peptide of factor XIII (a 37-residue extension) appear to be added for specialized functions after divergence of the tissue transglutaminase from their common lineage.

New thioester substrates for fibrinoligase (coagulation factor XIIIa) and for transglutaminase. Transfer of the fluorescently labeled acyl group to amines and alcohols.

Parameswaran KN, Lorand L
Biochemistry. 1981; 20: 3703-11

Hydrolysis of gamma:epsilon isopeptides by cytosolic transglutaminases and by coagulation factor XIIIa.

Parameswaran KN, Cheng XF, Chen EC, Velasco PT, Wilson JH, Lorand L
J Biol Chem. 1997; 272: 10311-7

Nepsilon-(gamma-glutamyl)lysine cross-links, connecting various peptide chain segments, are frequently the major products in transglutaminase-catalyzed reactions. We have now investigated the effectiveness of these enzymes for hydrolyzing the gamma:epsilon linkage. Branched compounds were synthesized, in which the backbone on the gamma-side of the cross-bridge was labeled with a fluorophor (5-(dimethylamino)-1-naphthalenesulfonyl or 2-aminobenzoyl) attached through an epsilon-aminocaproyl linker in the N-terminal position, and the other branch of the bridge was constructed with Lys methylamide or diaminopentane blocked by 2,4-dinitrophenyl at the Nalpha position. Hydrolysis of the cross-link could be followed in these internally quenched substrates by an increase in fluorescence. In addition to the thrombin and Ca2+-activated human coagulation Factor XIIIa, cytosolic transglutaminases from human red cells and from guinea pig liver were tested. All three enzymes were found to display good isopeptidase activities, with Km values of 10(-4) to 10(-5) M. Inhibitors of transamidation were effective in blocking the hydrolysis by the enzymes, indicating that expression of isopeptidase activity did not require unusual protein conformations. We suggest that transglutaminases may play a dynamic role in biology not only by promoting the formation but also the breaking of Nepsilon-(gamma-glutamyl)lysine isopeptides.

Partial purification and characterization of six transglutaminases from ordinary muscles of various fishes and marine invertebrates.

Nozawa H, Mamegoshi S, Seki N
Comp Biochem Physiol B Biochem Mol Biol. 1997; 118: 313-7

Six transglutaminases were prepared from ordinary muscles of scallop (Patinopecten yessoensis), botan shrimp (Pandalus nipponensis), squid (Todarodes pacificus), carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), and atka mackerel (Pleurogrammus azonus), and their physicochemical and enzymatic properties were compared with each other. The Km value of carp transglutaminase for monodansyl cadaverine, a kind of primary amines, was 0.33 mM, while the other enzymes had Km values of 0.01-0.03 mM. The Km, values for succinylated casein of scallop, botan shrimp, squid, carp, rainbow trout, and atka mackerel enzymes were 1.2, 0.3, 1.8, 0.3, 0.2, and 0.1 mg/ml, respectively. In the presence of 0.5 M NaCl, the activities of scallop, botan shrimp, and squid transglutaminases were further enhanced about 11-, 2-, and 6-fold, respectively. There was no effect of NaCl on the activities of fish enzymes. These increment in the activities were dependent of NaCl concentrations and could be exhibited by using KCl instead of NaCl. To date there are no reports on types of transglutaminases whose activities are stimulated by salts. These findings suggest there exist a novel type of TGase in marine invertebrate muscles in which the osmotic pressure is isotonic to sea water.

Crystal structure of red sea bream transglutaminase.

Noguchi K, Ishikawa K, Yokoyama Ki, Ohtsuka T, Nio N, Suzuki E
J Biol Chem. 2001; 276: 12055-9

The crystal structure of the tissue-type transglutaminase from red sea bream liver (fish-derived transglutaminase, FTG) has been determined at 2.5-A resolution using the molecular replacement method, based on the crystal structure of human blood coagulation factor XIII, which is a transglutaminase zymogen. The model contains 666 residues of a total of 695 residues, 382 water molecules, and 1 sulfate ion. FTG consists of four domains, and its overall and active site structures are similar to those of human factor XIII. However, significant structural differences are observed in both the acyl donor and acyl acceptor binding sites, which account for the difference in substrate preferences. The active site of the enzyme is inaccessible to the solvent, because the catalytic Cys-272 hydrogen-bonds to Tyr-515, which is thought to be displaced upon acyl donor binding to FTG. It is postulated that the binding of an inappropriate substrate to FTG would lead to inactivation of the enzyme because of the formation of a new disulfide bridge between Cys-272 and the adjacent Cys-333 immediately after the displacement of Tyr-515. Considering the mutational studies previously reported on the tissue-type transglutaminases, we propose that Cys-333 and Tyr-515 are important in strictly controlling the enzymatic activity of FTG.

Platelet factor XIII becomes active without the release of activation peptide during platelet activation.

Muszbek L, Polgar J, Boda Z
Thromb Haemost. 1993; 69: 282-5

The potentially active A subunit of factor XIII of blood coagulation has also been detected in platelets and monocytes/macrophages through the exact function of this cellular protransglutaminase has not yet been elucidated. In physiological conditions the first step in the activation of plasma factor XIII is the removal of an activation peptide from the N-terminal end of subunit A by thrombin. The A subunit then, in the presence of Ca2+, dissociates from the inhibitory B subunit and assumes an active conformation. Cellular factor XIII, which lacks B subunit, can be proteolytically activated in vitro by thrombin and the intracellular Ca2+ sensitive protease, calpain, in the same way as plasma factor XIII subunit A, and calpain has been suggested as the intracellular protease involved in the activation of cellular factor XIII in platelets. In the present experiments it was shown by SDS PAGE that during long-term stimulation of platelets with thrombin nondisulfide-crosslinked high M(r) protein polymers not penetrating the concentrating gel were formed. The lack of these polymers in thrombin-stimulated factor XIII deficient platelets clearly indicated that their formation in normal platelets was due to factor XIII that became active during platelet activation. However, no release of the activation peptide could be detected by Western blotting during this process. Similarly, no proteolytic cleavage of factor XIII was detectable when platelets were stimulated by Ca2+ ionophore through this stimulus activated calpain as it was clearly demonstrated by the breakdown of major intracellular calpain substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of cellular factor XIII into an active zymogen transglutaminase in thrombin-stimulated platelets.

Muszbek L, Haramura G, Polgar J
Thromb Haemost. 1995; 73: 702-5

The cellular form of blood coagulation factor XIII (FXIII) is present in platelets, monocytes and macrophages. During long-term stimulation of platelets by thrombin cellular FXIII becomes activated and cross-links proteins, however, the mechanism of its activation has not been elucidated. It was shown that, contrary to plasma FXIII, the intracellular activation of platelet FXIII does not involve proteolysis. FXIII remained intact in thrombin-activated platelets, i.e., the activation peptide was not removed from the molecule. Part of the zymogen FXIII molecules, however, assumed an active configuration as was demonstrated both by the measurement of transglutaminase activity and by active-site-SH titration. These findings clearly indicate that during platelet activation, when intracellular Ca2+ concentration is raised, a slow non-proteolytic transformation of FXIII zymogen into an active transglutaminase occurs.

Role of tissue transglutaminase in celiac disease.

Molberg O, McAdam SN, Sollid LM
J Pediatr Gastroenterol Nutr. 2000; 30: 232-40

Coagulation factor XIIIa undergoes a conformational change evoked by glutamine substrate. Studies on kinetics of inhibition and binding of XIIIA by a cross-reacting antifibrinogen antibody.

Mitkevich OV, Shainoff JR, DiBello PM, Yee VC, Teller DC, Smejkal GB, Bishop PD, Kolotushkina IS, Fickenscher K, Samokhin GP
J Biol Chem. 1998; 273: 14387-91

Coagulation factor XIIIa, plasma transglutaminase (endo-gamma-glutamine:epsilon-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen Aalpha-chain segment 529-539 was previously observed from analysis of end-stage plasma clots to block fibrin alpha-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit gamma-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFTNPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope Aalpha-(529-540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.

'Tissue' transglutaminase in cell death: a downstream or a multifunctional upstream effector?

Melino G, Piacentini M
FEBS Lett. 1998; 430: 59-63

Apoptotic cells show morphological modifications which occur as the result of complex molecular mechanisms involving several proteins including 'tissue' transglutaminase (tTG). Although tTG was originally thought to be responsible for the protein crosslinks which prevent the leakage of intracellular components, thereby reducing inflammation and autoimmunity, recent evidence indicates that tTG is a multifunctional enzyme involved in the complex upstream regulation of the apoptotic machinery: (i) it functions as a GTP-binding protein to transduce signals; (ii) it binds/crosslinks only specific cytosolic and nuclear substrates, suggesting highly specific actions, e.g. on intermediate filaments and in cell cycle control; (iii) it is finely tuned by Ca2+, GTP, S-nitrosylation, polyamines. In light of these recent discoveries, the role of tTG in the regulation of the crucial balance between survival and death is clearly complex.

Determinants of substrate specificity for factor XIII.

McDonagh J, Fukue H
Semin Thromb Hemost. 1996; 22: 369-76

Plasma factor XIIIa (A*2) is a regulator in balancing the opposing coagulation and fibrinolytic processes. Its enzymatic activity is to catalyze epsilon-(gamma-glutamyl)lysyl bonds between certain substrate molecules to link them by strong bonds. The primary physiological substrates are crosslinks between the gamma and alpha chains of fibrin that produce gamma-gamma-dimer and alpha-polymer, between alpha 2-plasmin inhibitor (alpha 2-PI) and alpha chains of fibrin, and between fibronectin and fibrin. We have characterized a unique factor XIII antibody that is specific for the middle 54-kDa section of A*2. It does not react with the zymogen (A2) or the inactive intermediate (A'2), and it does not inhibit the active center, as do most patient antibodies to factor XIII. This antibody inhibits the formation of A*2-fibrin complexes. Because of this specificity, the antibody was used to study other substrate interactions. It inhibited formation of fibronectin-factor XIIIa complexes, similarly to fibrin, and there was very little crosslinking of fibronectin to a fibrin clot. However, the amount of alpha 2-PI crosslinked to a fibrin clot was normal. It was concluded that this antibody interferes with exosite binding of fibrin and fibronectin interferes with exosite binding of fibrin and fibronectin in a similar way, while at least one critical exosite binding domain for alpha 2-PI is different from those of the other two substrates. Furthermore, with this antibody, it was shown that both alpha 2-PI-alpha chain crosslinking and alpha-polymer formation are necessary to normalize the rate of fibrinolysis.

A double-headed Gly-Pro-Arg-Pro ligand mimics the functions of the E domain of fibrin for promoting the end-to-end crosslinking of gamma chains by factor XIIIa.

Lorand L, Parameswaran KN, Murthy SN
Proc Natl Acad Sci U S A. 1998; 95: 537-41

The E domain of fibrinogen represents the central region of the protein that, after the removal of fibrinopeptides from the N-termini of its alpha chains by thrombin, orders the noncovalent assembly of fibrin units into a half-staggered array. This structural organization is accomplished purely through noncovalent binding between the E domain of one molecule and the distal D domains of two others. The process of assembly has a physiologically important up-regulatory effect on the next enzymatic phase of blood coagulation, which is the factor XIIIa-catalyzed end-to-end ligation of the gamma chains at the D domains of the protein. Fibrin assembly, as well as the acceleration of the factor XIIIa reaction, could be prevented by Gly-Pro-Arg-Pro, a homologue of the natural sequence of amino acids at the N termini of alpha chains in the E domain. We have now succeeded with a simple double-headed ligand, bis(Gly-Pro-Arg-Pro-amido)polyethylene glycol, in fully replacing the regulatory functions of the large E domains of the native protein.

Neurodegenerative diseases and transglutaminase.

Lorand L
Proc Natl Acad Sci U S A. 1996; 93: 14310-3

Synthesis and use of a substrate for the detection of isopeptidase activity.

Loewy AG, Blodgett JK, Blase FR, May MH
Anal Biochem. 1997; 246: 111-7

We have developed a substrate to assay for an isopeptidase, an enzyme capable of cleaving the Nepsilon-(gamma-glutamic) lysine bond which crosslinks polypeptide chains. This substrate consists of modified lysine (N-alpha-[3H]acetyl-l-lysine-N-methylamide or ALMA), linked by its epsilon-amino group to a gamma-carboxyl amide group of casein with guinea pig liver transglutaminase or Factor XIIIa. We used this substrate to demonstrate the release of [3H]ALMA from [3H]ALMA-casein in a culture medium of Bacillus cereus and in brain homogenates of 12- to 14-day-old embryonic chicks. The prokaryotic and the eukaryotic enzymes resemble each other in that both are activated by Ca2+ or Mg2+ and by alkaline phosphatase and both are inhibited by ATP. The [3H]ALMA-casein is a sensitive substrate able to measure reliably specific activities as low as 10(-8) micromol of [3H]ALMA/min/microg protein. The special advantage of this substrate is that the initial rate of ALMA-casein cleavage is not affected significantly by the levels of protease contaminants we have encountered. We were able to rule out alternative mechanisms such as gamma-glutamyl transpeptidase, gamma-glutamyl cyclotransferase, and the reversal of transglutaminase. We conclude that an isopeptidase mechanism most plausibly accounts for the ALMA release.

Site-directed mutagenesis of the calcium-binding site of blood coagulation factor XIIIa.

Lai TS, Slaughter TF, Peoples KA, Greenberg CS
J Biol Chem. 1999; 274: 24953-8

Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalently ligates fibrin molecules during blood coagulation. X-ray crystallography studies identified a major calcium-binding site involving Asp(438), Ala(457), Glu(485), and Glu(490). We mutated two glutamic acid residues (Glu(485) and Glu(490)) and three aspartic acid residues (Asp(472), Asp(476), and Asp(479)) that are in close proximity. Alanine substitution mutants of these residues were constructed, expressed, and purified from Escherichia coli. The K(act) values for calcium ions increased by 3-, 8-, and 21-fold for E485A, E490A, and E485A,E490A, respectively. In addition, susceptibility to proteolysis was increased by 4-, 9-, and 10-fold for E485A, E490A, and E485A,E490A, respectively. Aspartic acids 472, 476, and 479 are not involved directly in calcium binding since the K(act) values were not changed by mutagenesis. However, Asp(476) and Asp(479) are involved in regulating the conformation for exposure of the secondary thrombin cleavage site. This study provides biochemical evidence that Glu(485) and Glu(490) are Ca(2+)-binding ligands that regulate catalysis. The binding of calcium ion to this site protects the molecule from proteolysis. Furthermore, Asp(476) and Asp(479) play a role in modulating calcium-dependent conformational changes that cause factor XIIIa to switch from a protease-sensitive to a protease-resistant molecule.

Sphingosylphosphocholine reduces the calcium ion requirement for activating tissue transglutaminase.

Lai TS, Bielawska A, Peoples KA, Hannun YA, Greenberg CS
J Biol Chem. 1997; 272: 16295-300

Tissue transglutaminase (tTG) catalyzes a Ca2+-dependent transglutaminase reaction resulting in the formation of gamma-glutamyl-epsilon-lysine bonds and is activated during apoptosis to catalyze the formation of apoptotic body. We investigate whether lipids that are membrane components and involved in cell signaling could modify the Ca2+-dependent activation of tTG. We found that sphingosylphosphocholine (lyso-SM) was the only lipid to activate transglutaminase at low Ca2+ concentrations. In the presence of lyso-SM (125 microM), transglutaminase was detectable at 10 microM Ca2+, whereas in the absence of lyso-SM, similar activity was obtained at 160 microM Ca2+. Furthermore, in the presence of lipid vesicles lyso-SM retained the ability to enhance the Ca2+-dependent activation of tTG. Lyso-SM did not significantly change the Km for the glutamyl and primary amine substrates. However, the Kact for Ca2+ was reduced from 300 microM to 90 microM. Structure-function studies of lyso-SM analogs indicate that phosphocholine group on C1, the free amino group at C2 and a C4-C5 double bond are critical for the activation of transglutaminase activity. This is the first demonstration that a specific sphingolipid could enhance the activity of tTG and could play a role in vivo in activation of the tTG at physiologic Ca2+ levels.

The structure of the transglutaminase 1 enzyme. Deletion cloning reveals domains that regulate its specific activity and substrate specificity.

Kim SY, Kim IG, Chung SI, Steinert PM
J Biol Chem. 1994; 269: 27979-86

Transglutaminase 1 (TGase1) is one of three known enzymes involved in terminal differentiation in stratified squamous epithelia, possibly in the formation of a cornified cell envelope. Because the intact enzyme is particularly difficult to isolate in quantity from keratinocytes for characterization, comparatively little is known about its properties. We have expressed the full-length as well as a series of deletion forms of this enzyme in a bacterial system and analyzed their enzymatic properties. The specific activity of the full-length enzyme isolated and purified from the bacterial lysate was comparable to that of the native enzyme of keratinocytes. Analysis of several deletion constructs demonstrated that removal of the first 60-109 residues, which include sequences involved in membrane association, results in upwards of a 10-fold increase in the specific activity. Deletions beyond residue 109, into sequences conserved within the TGase family of proteins, result in loss of activity. Similarly, as many as 240 residues can be removed from its carboxyl-terminal end before activity is lost. Thus, a molecule of 466 residues, containing virtually only the conserved core sequences of TGases, retains a specific activity comparable to the intact enzyme. In addition, the various deletion forms display wide variations in substrate specificity toward a series of synthetic peptide substrates, designed from possible target TGase1 substrate proteins of epithelia. The data show that sequences between residues 62 and 92 are important in defining the substrate specificity of the TGase1 enzyme system. Furthermore, it may now be possible to design an enzyme with a defined substrate specificity. Together, these data suggest TGase1 has recruited additional sequences on its amino terminus in relation to other members of the TGase family, which have the net effect of permitting sequestration onto membranes, changing its specific activity and modifying its likely substrate specificities.

Structure and organization of the human transglutaminase 3 gene: evolutionary relationship to the transglutaminase family.

Kim IG, Lee SC, Lee JH, Yang JM, Chung SI, Steinert PM
J Invest Dermatol. 1994; 103: 137-42

The human haploid genome contains a family of at least five different transglutaminases that are differentially expressed in time- and tissue-specific ways. Of these, transglutaminase 3 (TGase3) is unusual in that it is a pro-enzyme requiring activation by proteolysis. To date it is known to be expressed only in terminally differentiating epidermal and hair follicle keratinocytes. In this paper we show that it is encoded by a gene (TGM3) of 42.8 kbp containing 13 exons. In the course of isolation of genomic clones for the TGM3 gene, we also found clones encoding the widely expressed tissue or TGase2 enzyme, perhaps due to high degrees of sequence homology. The structure of the TGM2 gene has not yet been reported. Our incomplete data suggest its exon/intron organization is very similar to that of TGM3. Although the common intron splice points of all members of the transglutaminase gene family have been conserved, the TGM3 and TGM2 genes, and the gene for the subplasma membrane transglutaminase-like protein band 4.2, lack two introns found in the TGM1 and factor XIIIa genes, and the exact intron splice point of another intron is shifted with respect to that of the TGM1 and factor XIIIa genes. Based on sequence homologies and gene structures, the data support a phylogenic tree in which the TGM2 and TGM3 genes belong on a branch distinct from other transglutaminases.

The deduced sequence of the novel protransglutaminase E (TGase3) of human and mouse.

Kim IG, Gorman JJ, Park SC, Chung SI, Steinert PM
J Biol Chem. 1993; 268: 12682-90

At least three transglutaminases are involved in terminal differentiation events in the epidermis and its derivatives, such as the hair follicle, presumably in cross-linking structural proteins and in the formation of the cornified cell envelope. Of these, only the transglutaminase 3 is a proenzyme, requiring activation by proteolytic cleavage, and is the least understood. Using oligonucleotides designed from the amino acid sequences of peptides of the guinea pig enzyme, we amplified mRNA and deduced the complete amino acid sequences of the mouse and human protransglutaminase 3 enzymes. Both proteins contain 692 amino acids of molecular mass about 77 kDa. Following expression in yeast, the proenzymes encoded by the full-length cDNA clones are active enzymes and can be further activated 15-fold on treatment with dispase. Although these proteins share 38-53% identity to other members of the transglutaminase family, surprisingly, the mouse, human, and guinea pig enzymes have not been highly conserved and show only 50-75% identity to each other. Much of the sequence variation occurs in the vicinity of the proteolytic activation site which lies at the most flexible and hydrophilic region of the molecule and is flanked by a sequence of 12 residues that are absent from other transglutaminases. We suggest that cleavage of this exposed flexible hinge region promotes a conformational change in the protein to a more compact form, resulting in activation of the enzyme. Expression of mouse and human protransglutaminase 3 mRNAs is regulated by calcium, as for other late differentiation products of the epidermis, suggesting that this enzyme is responsible for the later stages of cell envelope formation in the epidermis and hair follicle.

Identification and characterization of two missense mutations causing factor XIIIA deficiency.

Kangsadalampai S, Chelvanayagam G, Baker R, Tiedemann K, Kuperan P, Board PG
Br J Haematol. 1999; 104: 37-43

In this study, two amino acid substitutions, Arg260His and Val414Phe, have been identified in the factor XIIIA subunits of factor XIII deficient patients of Syrian and Indian descent, respectively. To confirm the deleterious effects of these substitutions, both variant sequences have been engineered into cDNA clones and the mutant enzymes expressed in yeast. Determination of the transglutaminase activity and immuno detection of the mutant enzymes together with mRNA hybridization revealed that the mutations dramatically reduce both the catalytic activity and the level of enzyme expressed in yeast. The mutations Arg260His and Val414Phe occur within the 'core' domain of the enzyme. Computer modelling of the mutant enzymes reveals that the substitution of the Arg260 by His results in the loss of a conserved electrostatic interaction whereas the effect of the Val414Phe substitution is a consequence of the large increase in side-chain volume. Although both mutations do not effect the active site directly, they are predicted to reduce the stability of the enzyme. The effects of these two amino acid substitutions on enzyme expression and three-dimensional structure strongly confirm that residues which are located outside of the active site can have a significant effect on protein stability and function.

The fibronectin-binding domain of transglutaminase.

Jeong JM, Murthy SN, Radek JT, Lorand L
J Biol Chem. 1995; 270: 5654-8

Guinea pig liver transglutaminase (EC 2.3.2.13) displays a Ca(2+)-independent binding (Ka = 10(7) M-1) to the same gelatin-binding domain of human plasma fibronectin that is known to form a very tight complex with the human red cell enzyme. The fibronectin-combining site of the liver transglutaminase was investigated by testing fragments obtained from the parent protein by controlled digestion with endoproteinase Lys-C. Overlay assays, probed with anti-fibronectin antibody, revealed that the fibronectin binding ability of the transglutaminase was encoded in a linear sequence in its 28-kDa N-terminal domain. Removal of the first 7 residues by further digestion of the purified 28-kDa material with endoproteinase Glu-C generated a 27-kDa fragment that, however, showed no binding activity. Thus, residues 1-7 in the liver enzyme seem to be of particular importance for influencing its ability to bind to fibronectin.

The comparative ability of plasma and tissue transglutaminases to use collagen as a substrate.

Jelenska MM, Fesus L, Kopec M
Biochim Biophys Acta. 1980; 616: 167-78

Heat denatured type I and type III calf skin collagen were found to be substrates for guinea pig liver transglutaminase (R-glutaminyl-peptide:amine gamma-glutamyl-yltransferase, EC 2.3.2.13) but not for active plasma factor XIII (factor XIIIa). Liver transglutaminase was shown to catalyse incorporation of 14C-putrescine into subunits of denatured collagen of both types, cross-linking of the latter into high molecular weight polymers and their co-cross-linking to fibrin and fibrinogen. Factor XIIIa is inactive in these respects. None of these reactions was catalysed by liver transglutaminase and plasma factor XIIIa when nondenatured collagens both soluble or in the forms of reconstituted fibrils served as substrates. Some cross-linking of cleavage products of collagen type I (obtained by treatment with collagenase from human neutrophiles) was induced by liver transglutaminase and factor XIIIa. The results indicate that although appropriate glutamine and lysine residues for a epsilon-(gamma-glutamine) lysine cross-linked formation are present in collagen, the native conformation of collagen prevents the action of liver transglutaminase and factor XIIIa.

[Various physiological functions suggested for protein cross-linking enzyme, transglutaminase]

Ikura K
Seikagaku. 1997; 69: 416-20

Consequences of seven novel mutations on the expression and structure of keratinocyte transglutaminase.

Huber M, Yee VC, Burri N, Vikerfors E, Lavrijsen AP, Paller AS, Hohl D
J Biol Chem. 1997; 272: 21018-26

We report the molecular characterization of seven new keratinocyte transglutaminase mutations (R315C, S358R, V379L, G473S, R687C, deletion Delta679-696, R127Stop) found in lamellar ichthyosis patients. Arg-315, Ser-358, Val-379, and Gly-473 are highly conserved residues in transglutaminases while Arg-687 and Delta679-696 are not. All mutations strongly decreased transglutaminase activity and protein levels. The mutation R127Stop diminished the amount of mRNA. Structural analysis of these mutations based on the factor XIII A-subunit crystal structure demonstrated that Arg-315, Ser-358, Val-379, and Gly-473 are located in the catalytic core domain, and Arg-687 and the deletion are in the beta-barrel domains. The side chains of amino acids Arg-315, Ser-358, and Gly-473 make ionic and hydrogen bonds important for folding and structural stability of the enzyme but are not directly involved in catalysis. Val-379 is two amino acids away from the active site cysteine, and its change into leucine disturbs the active site structure. The decreased activity and protein level after expression of the R687C and Delta679-696 TGK cDNA in TGK negative keratinocytes excluded that they are polymorphisms. These results identify important amino acids in the central core domain of transglutaminases and show that the C-terminal end influences the structural and functional integrity of TGK.

Analysis of factor XIII substrate specificity using recombinant human factor XIII and tissue transglutaminase chimeras.

Hettasch JM, Peoples KA, Greenberg CS
J Biol Chem. 1997; 272: 25149-56

Human factor XIII (FXIII) and tissue transglutaminase (tTG) are homologous proteins. FXIII requires thrombin for activation and cross-links the gamma chains of fibrin(ogen) more efficiently than the Aalpha chains. On the other hand, tTG is thrombin-independent and forms predominantly Aalpha and Aalpha-gamma chain complexes. Previous work from this laboratory demonstrated that amino acid residues within exon 7 of FXIII were important for catalysis (Hettasch, J. M., and Greenberg, C. S. (1994) J. Biol. Chem. 269, 28309-28313). To determine to what extent the primary amino acid sequence within exon 7 defines substrate specificity, exon 7 of FXIII was replaced with the corresponding exon of tTG using gene splicing by overlap extension. Other work from this laboratory (Achyuthan, K. E., Slaughter, T. F., Santiago, M. A., Enghild, J. J., and Greenberg, C. S. (1993) J. Biol. Chem. 268, 21284-21292) using synthetic peptides identified two other domains that might play a role in substrate recognition (located in exons 3 and 5). Therefore, recombinant chimeras of FXIII/tTG were also created in which these two exons were exchanged. FXIII, tTG, and chimeras 3, 5, and 7 were expressed in Escherichia coli, purified, and the nature of the fibrin cross-linking pattern of these five proteins was determined by immunoblot analysis. FXIII preferentially formed the gamma-gamma dimer, whereas tTG formed Aalpha-gamma complexes. Chimera 7 formed Aalpha-gamma complexes that resembled the cross-linking pattern of tTG. This finding demonstrates that the primary amino acid sequence of exon 7 of tTG confers some of the specificity for the Aalpha and Aalpha-gamma cross-link pattern characteristic of tTG. Chimera 5 exhibited reduced cross-linking activity (50% of FXIII activity) but still retained preference for formation of the gamma-gamma dimer, whereas chimera 3 was not active. In conclusion, exchanging the primary amino acid sequence of the active site exon of human FXIII with that of human tTG modifies the enzyme such that the fibrin cross-linking pattern more closely resembles that of tTG (Aalpha and Aalpha-gamma complexes) instead of FXIII (gamma-gamma dimers).

Analysis of the catalytic activity of human factor XIIIa by site-directed mutagenesis.

Hettasch JM, Greenberg CS
J Biol Chem. 1994; 269: 28309-13

Factor XIIIa (FXIIIa) stabilizes fibrin clots by covalently cross-linking fibrin molecules. The purpose of this study was to determine the amino acid requirements at the active site of FXIIIa for catalysis. We selected amino acids 310-317 (Arg-Tyr-Gly-Gln-Cys-Trp-Val-Phe) in the human FXIII A-chain sequence for analysis based on the high degree of sequence homology among the different transglutaminases. We converted each amino acid in this region to Ala by site-directed mutagenesis. These recombinant FXIII A-chain mutants were expressed in Escherichia coli using the pTrc99A expression vector. FXIIIa activity was assessed by measuring the incorporation of 5-(biotinamido)pentylamine into N,N'-dimethylcasein in a solid-phase microtiter plate assay. The Cys-314-->Ala mutation yielded a recombinant protein with no FXIIIa activity. We also found that changing Gly-312 and Val-316 to Ala resulted in 22 and 65% decreases in activity, respectively. The other five mutations near the active-site Cys resulted in FXIIIa molecules in which the activity was reduced > 95%. The mechanism of SH protease catalysis is similar to transglutaminase catalysis in that both form thioester intermediates. His and Asp residues may stabilize this enzyme-substrate intermediate. Therefore, we performed site-directed mutagenesis on several His residues (His-342, His-373, and His-450) as well as Asp-396 in human FXIII. We found that changing His-342 to Ala reduced catalytic activity by 85%, while the His-373-->Ala mutant had no activity. In contrast, changing His-450 to Ala reduced FXIIIa activity by only 15%. We also examined the activity of all the mutants in a fibrin cross-linking assay. Four of the mutations (Phe-317-->Ala, Tyr-315-->Ala, Gln-313-->Ala, and Asp-396-->Ala), in which the activity toward the small primary amine was reduced by > 95%, were still capable of cross-linking the gamma-chain of fibrin. Even though these four mutants produced gamma-gamma dimers, they were not capable of forming higher molecular weight cross-linked products. Finally, we found that the binding of all the mutants to fibrin was similar to that of wild-type FXIIIa. In conclusion, we demonstrated that changing the specific amino acids Arg-310-Phe-317 to Ala substantially reduced FXIIIa activity. In addition, full catalytic activity was dependent on His-342, His-373, and Asp-396. These findings provide new insights into the catalytic mechanism of FXIIIa.

Bovine histidine-rich glycoprotein is a substrate for bovine plasma factor XIIIa.

Halkier T, Andersen H, Vestergaard A, Magnusson S
Biochem Biophys Res Commun. 1994; 200: 78-82

Histidine-rich glycoprotein was purified from bovine plasma and the identity of the protein confirmed through amino acid sequencing. Activated bovine factor XIIIa catalyzed the incorporation of 1 nmol of 1,4-[14C]putrescine into 1 nmol of bovine histidine-rich glycoprotein, showing that histidine-rich glycoprotein has the ability to participate in transglutaminase-catalyzed reactions in vivo.

Substrate requirements for transglutaminases. Influence of the amino acid residue preceding the amine donor lysine in a native protein.

Grootjans JJ, Groenen PJ, de Jong WW
J Biol Chem. 1995; 270: 22855-8

Thirteen recombinant alpha A-crystallin mutants were constructed that differed in the type of amino acid residue directly preceding the sole amine donor lysine for transglutaminases in this protein. The capacity of these mutants to be cross-linked to amine acceptor substrates by tissue transglutaminase and factor XIII was assessed. Two different biotinylated glutamine-containing oligopeptides were used as amine acceptor probes. It appears that the type of residue preceding the amine donor lysine has a considerable influence on the substrate potential of alpha A-crystallin for transglutaminases. This influence shows qualitatively similar trends for tissue transglutaminase and factor XIII and is irrespective of the amine acceptor probe. In general, glycine or aspartic acid before the amine donor lysine has the strongest adverse effects on substrate reactivity, and proline, histidine, and tryptophan are less favorable. Valine, arginine, and phenylalanine, and to a more variable or somewhat lesser extent also serine, alanine, leucine, tyrosine, and asparagine, have an enhancing effect. This pattern of preference is largely in agreement with that observed for the limited number of characterized amine donor lysines in protein substrates for transglutaminases. It can be concluded that tissue transglutaminase and factor XIII have a rather broad yet clearly differentiated tolerance with respect to the residue preceding the amine donor lysine substrate in native proteins.

Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues.

Greenberg CS, Birckbichler PJ, Rice RH
FASEB J. 1991; 5: 3071-7

Transglutaminases catalyze the posttranslational modification of proteins by transamidation of available glutamine residues. This action results primarily in the formation of epsilon-(gamma-glutamyl)lysine cross-links but includes the incorporation of polyamines into suitable protein substrates as well. The covalent isopeptide crosslink is stable and resistant to proteolysis, thereby increasing the resistance of tissue to chemical, enzymatic, and mechanical disruption. The plasma transglutaminase, factor XIIIa, is formed at sites of blood coagulation and impedes blood loss by stabilizing the fibrin clot. The squamous epithelium constituting the protective callus layer of skin is formed by the action of keratinocyte transglutaminase (TGK) and epidermal transglutaminase (TGE). The tissue transglutaminase (TGC) is a cytoplasmic enzyme present in many cells including those in the blood vessel wall. TGC function is unknown, although it could function to stabilize intra- and extra-cellular molecules in a wide variety of physiologic or pathologic processes. The amino acid sequences of factor XIII, TGC, and TGK establish them as a homologous gene family and also reveal a striking homology to the erythrocyte membrane protein, band 4.2. This review summarizes the current information on structures, functions, and evolution of the most prominent members of this gene family.

Structural features of glutamine substrates for transglutaminases. Role of extended interactions in the specificity of human plasma factor XIIIa and of the guinea pig liver enzyme.

Gorman JJ, Folk JE
J Biol Chem. 1984; 259: 9007-10

Amino acid residues at several locations in close primary vicinity to a substrate glutamine residue have been recognized as important determinants for the specificities of human plasma factor XIIIa and guinea pig liver transglutaminase (Gorman, J. J., and Folk, J. E. (1981) J. Biol. Chem. 256, 2712-2715). The present studies measure the influence on transglutaminase specificity of some changes in amino acid side chains in a small synthetic glutamine peptide amide, Leu-Gly-Leu-Gly-Gln-Gly-Lys-Val-Leu-GlyNH2, which was designed to contain most of the known elements needed for enzyme recognition. The results are in agreement with previous findings and show that full catalytic activity of each enzyme may be retained upon replacement of the lysine residue by certain other amino acid residues. Evidence is provided that serine in place of glycine at one or more positions causes a significant increase in specificity with factor XIIIa, but not with liver enzyme. The effective substrate property for factor XIIIa seen with the model peptide amide is lost upon reversal of the sequence Val-Leu. This is not the case with the liver enzyme even though replacement of either of these amino acids by alanine causes a pronounced loss in activity with this enzyme. These differences and the effects of various other substitutions in the model peptide amide on the enzymes' specificities points up the relatively stringent structural requirements of factor XIIIa and the rather broad requirements for liver transglutaminase.

Cross induced coagulations between human and crustacean clotting factors. Considerations on the clotting processes.

Ghidalia W, Vendrely R, Montmory C, Coirault Y
Comp Biochem Physiol A. 1982; 72: 741-5

1. Cross induced coagulations show that human factor XIII and crustacean coagulin are to some extent functionally equivalent and may be substituted for each other. 2. In crustacea, fibrinogen and coagulin appear as in situ activated products since they are both able to react with non-activated human clotting factors. 3. The coagulin catalyzed transamidation which stabilizes the clot and renders it insoluble in 1-5% monochloroacetic acid solutions seems to be the basic reaction of the clotting process in the animals in which coagulation occurs. 4. The possibility of a two step clotting in crustacea is discussed.

Mechanism and basis for specificity of transglutaminase-catalyzed epsilon-(gamma-glutamyl) lysine bond formation.

Folk JE
Adv Enzymol Relat Areas Mol Biol. 1983; 54: 1-56

Searching for the function of tissue transglutaminase: its possible involvement in the biochemical pathway of programmed cell death.

Fesus L, Thomazy V
Adv Exp Med Biol. 1988; 231: 119-34

Although several details are still missing, the biological role of two of the three well characterized transglutaminases in mammals, namely blood coagulation factor XIII and keratinocyte transglutaminase, is established. The function of the third one called the tissue type is still an enigma. Its constant localization in endothelial and smooth muscle cells of all organs, in heart muscle, in medullary interstitial and mesangial cells of kidney, and its induction in a number of other cell types under a variety of conditions suggest multiple functions. According to our results its participation in the biochemical pathway leading to programmed cell death (apoptosis), a basic cellular phenomenon of physiological significance, may be one of these functions.

Platelet-associated factor XIII in platelet activation, adhesion, and clot stabilization.

Devine DV, Bishop PD
Semin Thromb Hemost. 1996; 22: 409-13

Platelet-associated factor XIII provides a means by which to promote clot stabilization and platelet interaction with proteins of the coagulation and fibrinolytic pathways. In addition to its intracellular role within the platelet cytoplasm, activated factor XIII will bind to the surface of activated platelets. These platelets then participate in cell-cell or cell-clot interactions, thereby increasing the local concentration of factor XIIIa. The platelet-associated factor XIIIa may increase the amount of crosslinking in a fibrin clot, thereby contributing to the aging of the clot and the reduction in the degree of platelet binding. Clot resistance to fibrinolysis is enhanced by platelet factor XIIIa-mediated crosslinking of alpha 2-antiplasmin to fibrin. The binding of factor XIIIa to the platelet surface requires the activation of the platelet fibrinogen receptor, glycoprotein IIb-IIIa. Thus, platelet-associated factor XIIIa may be used as a marker of in vivo platelet activation. Since half of the factor XIII present in blood is provided by the platelets, it is not surprising that this form of factor XIII plays an important role in hemostasis.

A continuous spectrophotometric linked enzyme assay for transglutaminase activity.

Day N, Keillor JW
Anal Biochem. 1999; 274: 141-4

Transglutaminases and their regulation: implications for polyamine metabolism.

Davies PJ, Chiocca EA, Basilion JP, Poddar S, Stein JP
Adv Exp Med Biol. 1988; 250: 391-401

Regulation of factor XIIIa generation by fibrinogen.

Curtis CG, Janus TJ, Credo RB, Lorand L
Ann N Y Acad Sci. 1983; 408: 567-76

Stimulation of phospholipases A2 by transglutaminases.

Cordella-Miele E, Miele L, Beninati S, Mukherjee AB
Adv Exp Med Biol. 1990; 279: 105-23

The structural basis for the regulation of tissue transglutaminase by calcium ions.

Casadio R, Polverini E, Mariani P, Spinozzi F, Carsughi F, Fontana A, Polverino de Laureto P, Matteucci G, Bergamini CM
Eur J Biochem. 1999; 262: 672-9

The role of calcium ions in the regulation of tissue transglutaminase is investigated by experimental approaches and computer modeling. A three-dimensional model of the transglutaminase is computed by homology building on crystallized human factor XIII and is used to interpret structural and functional results. The molecule is a prolate ellipsoid (6.2 x 4.2 x 11 nm) and comprises four domains, assembled pairwise into N-terminal and C-terminal regions. The active site is hidden in a cleft between these regions and is inaccessible to macromolecular substrates in the calcium-free form. Protein dynamics simulation indicates that these regions move apart upon addition of calcium ions, revealing the active site for catalysis. The protein dimensions are consistent with results obtained with small-angle neutron and X-ray scattering. The gyration radius of the protein (3 nm) increases in the presence of calcium ions (3.9 nm), but it is virtually unaffected in the presence of GTP, suggesting that only calcium ions can promote major structural changes in the native protein. Proteolysis of an exposed loop connecting the N-terminal and C-terminal regions is linearly correlated with enzyme inactivation and prevents the calcium-induced conformational changes.

Activation of transglutaminase during embryonic development.

Cariello L, Wilson J, Lorand L
Biochemistry. 1984; 23: 6843-50

Incorporation of [3H]putrescine into proteins was shown to increase markedly in sea urchin eggs upon fertilization. Emetine, an inhibitor of protein synthesis, had no effect on the rate of protein labeling. However, the reaction could be prevented by the addition of 2-[3-(diallylamino)-propionyl]benzothiophene, a noncompetitive inhibitor of transglutaminase, and also by dansylcadaverine, which is a substrate for transglutaminase. The inert N alpha-dimethyl analogue of dansylcadaverine had no influence. Considering the complexity of the incorporation of the [3H]putrescine tracer in this system, it was deemed essential to prove by rigorous analytical methods that the reaction was, indeed, consistent with a transglutaminase mechanism. gamma-Glutamyl[3H]putrescine could be recovered in 80-90% yield from the proteolytic digest of proteins from the 20-min fertilized cell. Another sign of the in vivo activity of transglutaminase was the isolation of substantial amounts of epsilon-(gamma-glutamyl)lysine from proteins of sea urchin embryo, yielding a frequency value for this cross-link as high as 1 mol/400 000 g of protein in the 32-cell-stage material.

Transglutaminase 1 mutations in lamellar ichthyosis. Loss of activity due to failure of activation by proteolytic processing.

Candi E, Melino G, Lahm A, Ceci R, Rossi A, Kim IG, Ciani B, Steinert PM
J Biol Chem. 1998; 273: 13693-702

Lamellar ichthyosis is a congenital recessive skin disorder characterized by generalized scaling and hyperkeratosis. It is caused by mutations in the TGM1 gene that encodes the transglutaminase 1 (TGase 1) enzyme, which is critical for the assembly of the cornified cell envelope in terminally differentiating keratinocytes. TGase 1 is a complex enzyme existing as both cytosolic and membrane-bound forms. Moreover, TGase 1 is proteolytically processed, and the major functionally active form consists of a membrane-bound 67/33/10-kDa complex with a myristoylated and palmitoylated amino-terminal 10-kDa membrane anchorage fragment. To understand better how point mutations, deletions, and truncations found in lamellar ichthyosis disease affect the structure and function of TGase 1, we have expressed in baculovirus and keratinocytes a number of reported TGase 1 mutants. The structural implications of these mutations were examined using a homology-derived three-dimensional model of TGase 1 generated from the known x-ray structure of the related coagulation factor XIIIa enzyme. The present studies demonstrate that loss of TGase 1 activity is not restricted to mutations that directly affect the enzymatic activity. We report a new class of mutations that impair the subsequent post-synthetic processing of the protein into its highly active functional forms.

Location of the major epsilon-(gamma-glutamyl)lysyl cross-linking site in transglutaminase-modified human plasminogen.

Bendixen E, Harpel PC, Sottrup-Jensen L
J Biol Chem. 1995; 270: 17929-33

Tissue and plasma transglutaminases cross-link human plasminogen into high molecular weight complexes (Bendixen, E., Borth, W., and Harpel, P. C. (1993) J. Biol. Chem. 268, 21962-21967). A major cross-linking site in plasminogen involved in the tissue transglutaminase-mediated polymerization process has been identified. The epsilon-(gamma-glutamyl)lysyl bridges of the polymer are formed between Lys-298 and Gln-322. Both the acyl donor Gln residue and the acyl acceptor Lys residue are located in the kringle 3 domain of plasminogen, i.e. cross-linking of plasminogen by tissue transglutaminase involves neither the catalytic domain nor the lysine-dependent binding sites of plasminogen. This study documents that kringle 3 contains a novel functional site with the potential to participate in transglutaminase-mediated cross-linking interactions with plasma, cell-surface, and extracellular proteins.

Subtyping of coagulation factor XIIIA.

Arnold G, Kloor D, Kompf J, Ritter H
Hum Hered. 1995; 45: 319-22

An extended polymorphism of the coagulation factor XIIIA can routinely be detected in human plasma samples and white cell lysates by isoelectric focusing in polyacrylamide gels containing 3 M urea in the pH range 5-8. Analyses of 184 families with 513 children confirmed the formal model proposed by Suzuki et al. [Am J Hum Genet 1988;43:170-174]. Four common alleles, F XIIIA*1A, 1B, 2A, 2B, at an autosomal locus control the expression of ten phenotypes. On the basis of the population sample from southwest Germany the frequencies of the common alleles F XIIIA*1A, 1B, 2A, 2B were calculated as 0.175, 0.609, 0.011, and 0.205, respectively.

Transglutaminases: protein cross-linking enzymes in tissues and body fluids.

Aeschlimann D, Paulsson M
Thromb Haemost. 1994; 71: 402-15

Tissue transglutaminase and factor XIII in cartilage and bone remodeling.

Aeschlimann D, Mosher D, Paulsson M
Semin Thromb Hemost. 1996; 22: 437-43

While it is well established that factor XIII functions in crosslinking of the fibrin clot during blood coagulation and in wound healing, the physiological role of tissue transglutaminase is still unclear. Recent studies suggest that the expression of tissue transglutaminase correlates with (terminal) differentiation of cells and that the enzyme may play a role in extracellular matrix remodeling. In cartilage, tissue transglutaminase expression is restricted to hypertrophic chondrocytes and the enzyme is externalized at a distinct step in the chondrocyte maturation program. Upon activation by Ca2+, the transglutaminase modifies matrix constituents in a way that might predispose the matrix for the subsequent mineralization. Crosslinks of the structure gamma-glutamyl-epsilon-lysine are also abundant in bone matrix, but the transglutaminase expressed by osteoblasts and presumably involved in crosslinking of newly formed osteoid is likely to be distinct from both tissue transglutaminase and factor XIII. Matrix proteins thought to be crosslinked by transglutaminases in cartilage and bone matrix include glycoproteins such as osteonectin, osteopontin, fibronectin, fibrillin, and collagens II, III, V, and XI. Expression of the A subunit of factor XIII is restricted to megakaryocytes in the bone marrow cavity, and factor XIIIa is abundant in platelets that probably provide the major source for factor XIII in plasma.

Factor XIIIa-derived peptides inhibit transglutaminase activity. Localization of substrate recognition sites.

Achyuthan KE, Slaughter TF, Santiago MA, Enghild JJ, Greenberg CS
J Biol Chem. 1993; 268: 21284-92

Factor XIIIa is a transglutaminase that catalyzes intermolecular gamma-glutamyl-epsilon-lysyl bonds between fibrin and other proteins involved in hemostasis. We synthesized 25 peptides from various regions of factor XIIIa and studied their effects on cross-linking fibrin, N,N'-dimethylcasein, or fibronectin. We found that two peptides, Asn72-Asp97 (peptide-4) and Asp190-Phe230 (peptide-7), inhibited factor XIIIa cross-linking of these substrates. The other peptides did not inhibit factor XIIIa activity. The inhibition of cross-linking was reversed by excess substrate, indicating that the peptides were interacting with fibrin and not factor XIIIa. The peptides were not pseudosubstrates since they were not cross-linked to fibrin. The peptides did not modify the primary amine binding site as increasing the primary amine concentration did not reverse inhibition. Peptides-4 and -7 also had no effect on exposure of the active site of factor XIIIa and no synergistic inhibitory effects were detected. Peptides-4 and -7 had no effect on factor XIIIa binding to fibrin suggesting that the binding sites and the substrate recognition sites were distinct. Synthetic peptides containing shorter amino acid sequences of peptide-4 were inactive. In contrast, the amino-terminal (Asp190-Lys199, Tyr194-Tyr204) and the carboxyl-terminal (Lys221-Phe230) portions of peptide-7 were 20-60-fold less inhibitory compared to intact peptide-7. Peptides-4 and -7 also inhibited guinea pig liver tissue transglutaminase from cross-linking fibrinogen, N,N'-dimethylcasein, and fibronectin. In conclusion, we have identified two regions outside the active site pocket which are important for substrate recognition in factor XIIIa and tissue transglutaminase.

Enhanced chemiluminescent assay for transglutaminases.

Achyuthan KE
Biotechniques. 1999; 26: 435-8