Peptide-bound Glutamine Residues Research Articles

Biocatalysis by Transglutaminases: A Review of Biotechnological Applications.

The biocatalytic activity of transglutaminases (TGs) leads to the synthesis of new covalent isopeptide bonds (crosslinks) between peptide-bound glutamine and lysine residues, but also the transamidation of primary amines to glutamine residues, which ultimately can result into protein polymerisation. Operating with a cysteine/histidine/aspartic acid (Cys/His/Asp) catalytic triad, TGs induce the post-translational modification of proteins at both physiological and pathological conditions (e.g., accumulation of matrices in tissue fibrosis). Because of the disparate biotechnological applications, this large family of protein-remodelling enzymes have stimulated an escalation of interest. In the past 50 years, both mammalian and microbial TGs polymerising activity has been exploited in the food industry for the improvement of aliments’ quality, texture, and nutritive value, other than to enhance the food appearance and increased marketability. At the same time, the ability of TGs to crosslink extracellular matrix proteins, like collagen, as well as synthetic biopolymers, has led to multiple applications in biomedicine, such as the production of biocompatible scaffolds and hydrogels for tissue engineering and drug delivery, or DNA-protein bio-conjugation and antibody functionalisation. Here, we summarise the most recent advances in the field, focusing on the utilisation of TGs-mediated protein multimerisation in biotechnological and bioengineering applications.

Transglutaminases - a review with special reference to microbial transglutaminase and its application in food processing

Some properties and applications of the transglutaminase (TG), with particular focus on TG derived from microorganisms (MTG), are described. TG catalyzes an acyltransfer reaction in which the γ-carboxyamide groups of pep tidebound glutamine residues are the acyl-donors. Most food proteins, such as legume globulins, wheat gluten and gliadin, egg yolk and egg white proteins, meat actins and myosins, gelatin, collagen, milk caseins, a-lactalbumin and /Mactoglobulin, could be crosslinked by TG. TG are present in an extremely broad spectrum of living organisms, such as humans, most advanced animals, birds, amphibians, fish, plants and microorganisms. Commercial TG has been merely obtained from animal tissues for decades. The limited supply and the complicated separation and purification procedure for obtaining tissue TG have resulted in an extremely high price of the enzyme, which hampers a wide application in food processing. MTG, mass-produced at low cost by fermentation, catalyses the cross-linking of most food proteins through the formation of c-(v-glutamyl) lysine bonds, in the same way as wellknown mammalian enzymes. However, MTG is quite unique from other mammalian TG, since it is totally independent of Ca + and has a relatively lower molecular weight. The results of many studies suggest that MTG has many potential applications in food processing. Food treated with MTG appeared to have an improved flavour, appearance and texture. In addition, this enzyme can increase shelf-life and reduce allergenicity of certain foods. Using additional components, such as sodium ceseinate, maltodextrine and starch, MTG can be customized for use in many other foods, even those with lower protein content. In this respect, MTG technology will be an essential tool for producing acceptable protein foods from non-animal proteins in the future.

Transglutaminases in Dysbiosis As Potential Environmental Drivers of Autoimmunity.

Protein-glutamine γ-glutamyltransferases (transglutaminases, Tgs) belong to the class of transferases. They catalyze the formation of an isopeptide bond between the acyl group at the end of the side chain of protein- or peptide-bound glutamine residues and the first order 𝜀-amine groups of protein- or peptide-bound lysine. The Tgs are considered to be universal protein cross-linkers, and they play an essential role in a number of human diseases. In this review, we discuss mainly the bacterial Tgs in terms of the functionality of the enzymes and a potential role they may play in bacterial survival. Since microbial transglutaminases (mTgs) are functionally similar to the human homologs, they may be involved in the human disease provocation. We suggest here a potential involvement of Tgs in the pathologies such as autoimmune diseases. In this hypothesis, the endogenous mTgs that are secreted by the gut microbiota, especially in a dysbiotic configuration, are potential drivers of systemic autoimmunity, via the enzymatic posttranslational modification of peptides in the gut lumen. These mTg activities directed toward cross-linking of naïve proteins can potentially generate neo-epitopes that are not only immunogenic but may also activate some immune response cascades leading to the pathological autoimmune processes.

Distribution of Transglutaminase in Pear Pollen Tubes in Relation to Cytoskeleton and Membrane Dynamics

Transglutaminases (TGases) are ubiquitous enzymes that take part in a variety of cellular functions. In the pollen tube, cytoplasmic TGases are likely to be involved in the incorporation of primary amines at selected peptide-bound glutamine residues of cytosolic proteins (including actin and tubulin), while cell wall-associated TGases are believed to regulate pollen tube growth. Using immunological probes, we identified TGases associated with different subcellular compartments (cytosol, membranes, and cell walls). Binding of cytosolic TGase to actin filaments was shown to be Ca(2+) dependent. The membrane TGase is likely associated with both Golgi-derived structures and the plasma membrane, suggesting a Golgi-based exocytotic delivery of TGase. Association of TGase with the plasma membrane was also confirmed by immunogold transmission electron microscopy. Immunolocalization of TGase indicated that the enzyme was present in the growing region of pollen tubes and that the enzyme colocalizes with cell wall markers. Bidimensional electrophoresis indicated that different TGase isoforms were present in distinct subcellular compartments, suggesting either different roles or different regulatory mechanisms of enzyme activity. The application of specific inhibitors showed that the distribution of TGase in different subcellular compartments was regulated by both membrane dynamics and cytoskeleton integrity, suggesting that delivery of TGase to the cell wall requires the transport of membranes along cytoskeleton filaments. Taken together, these data indicate that a cytoplasmic TGase interacts with the cytoskeleton, while a different TGase isoform, probably delivered via a membrane/cytoskeleton-based transport system, is secreted in the cell wall of pear (Pyrus communis) pollen tubes, where it might play a role in the regulation of apical growth.

Redox Regulation of Transglutaminase 2 Activity

Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types of inflammation and cell injury. The enzymatic activity of extracellular TG2 thus appears to be tightly regulated. As TG2 is known to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys(230), Cys(370), and Cys(371) that is involved in oxidative inactivation of TG2. Within this triad, Cys(370) was found to participate in disulfide bonds with both Cys(230) and its neighbor, Cys(371). Notably, Ca(2+) was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys(230) appears to promote oxidation and inactivation of TG2 by facilitating formation of Cys(370)-Cys(371) through formation of the Cys(230)-Cys(370) disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys(230) is unique for TG2, suggesting that this residue acts as an isoform-specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extracellular environment.

TG2 protects neuroblastoma cells against DNA-damage-induced stress, suppresses p53 activation

Tissue transglutaminase (TG2) is a multifunctional member of the transglutaminase (TGase) family (E.C.2.3.2.13), which catalyzes in a calcium-dependent reaction the formation of covalent bonds between the gamma-carboxamide groups of peptide-bound glutamine residues and various primary amines. Here, we investigated the role of TG2 in a response of the neuroblastoma SH-SY5Y cells to topoisomerase II inhibitor etoposide, known to trigger DNA-damage cell response. We found an early and transient (approximately 2 h) increase of the TG2 protein in SH-SY5Y cells treated with etoposide, along with the increase of phosphorylated and total levels of the p53 protein. Next, we showed that SH-SY5Y cells, which overexpress wild-type TG2 were significantly protected against etoposide-induced cell death. The TG2 protective effect was associated only with the transamidation active form of TG2, because overexpression the wild-type TG2, but not its transamidation inactive C277S form, resulted in a pronounced suppression of caspase-3 activity as well as p53 phosphorylation during the etoposide-induced stress. In addition, exacerbation of cell death with a significant increase in caspase-3 and p53 activation was observed in SH/anti-TG2 cells, in which expression of the endogenous TG2 protein has been greatly reduced by the antisense cDNA construct. Though the cell signaling and molecular mechanisms of the TG2-driven suppression of the cell death machinery remain to be investigated, our findings strongly suggest that TG2 plays an active role in the response of neuroblastoma cells to DNA-damage-induced stress by exerting a strong protective effect, likely by the suppression of p53 activation and p53-driven cell signaling events.

Molecular mechanism of the interaction between activated factor XIII and its glutamine donor peptide substrate.

Activated factor XIII (FXIII), a dimer of truncated A-subunits (FXIII-A2*), is a transglutaminase that crosslinks primary amines to peptide-bound glutamine residues. Because in the few natural substrates of FXIII-A2* no consensus sequence could be identified around the reactive glutamine, studying the interaction between individual substrates and FXIII-A2* is of primary importance. Most of the alpha2-plasmin inhibitor (alpha2PI) molecules become truncated by a plasma protease, and the truncated isoform (N1-alpha2PI) is an important substrate of FXIII-A2*. The crosslinking of N1-alpha2PI to fibrin plays a major role in protecting fibrin from fibrinolysis. We studied the interaction of FXIII-A2* with its dodecapeptide glutamine donor substrate, N1-alpha2PI(1-12), the sequence of which corresponds to the N-terminal sequence of N1-alpha2PI. Kinetic parameters for N1-alpha2PI(1-12) and for its truncated or synthetic mutants were determined by a spectrophotometric assay. The interaction of N1-alpha2PI(1-12) with FXIII-A2* was investigated by proton nuclear magnetic resonance (NMR) and saturating transfer difference (STD) NMR. Kinetic experiments with peptides in which the Asn1 residue was either truncated or replaced by alanine and proton NMR analysis of the FXIII-A2*-N1-alpha2PI(1-12) complex demonstrated that Asn1 is essential for effective enzyme-substrate interaction. Experiments with C-terminally truncated peptides proved that amino acids 7-12 are essential for the interaction of N1-alpha2PI(1-12) with the enzyme, and suggested the existence of a secondary binding site on FXIII-A2*. Hydrophobic residues, particularly Leu10 and the C-terminal Lys12, seemed to be especially important in this respect, and direct interaction between hydrophobic C-terminal residues and FXIII-A2* was demonstrated by STD NMR.

Synthetic substrates as amine donors and acceptors in microbial transglutaminase-catalysed reactions

Microbial transglutaminase (EC 2.3.2.13) (mTGase) catalyses a calcium-independent acyl-transfer reaction in which ɛ-(γ-glutamyl)lysine bonds are formed using the γ-carboxyamide groups of peptide-bound glutamine residues and the amino group of lysine side-chains. Here we present a comparative study on alternative lysine and glutamine substitutes in mTGase catalysis. A homologous series of ω-amino acids, serving as lysine substitutes, was incorporated into carbobenzoxy- l-glutaminylglycine (CBZ-Gln-Gly). The rate constants and particular conversion rates increased with increasing chain length. As for the glutamine substitutes, adipic diamide, glutaric monoamide, and glutaric diamide were converted with monodansylcadaverine (DNS-cadaverine) under mTGase catalysis. For the synthetic glutamine substitutes, the substrates of natural chain length, glutaric mono- and diamide, are better converted than the longer adipic diamide indicating that the window of opportunity seems to be smaller. Synthetic substrates, serving as amine acceptors, offer new opportunities in the field of transglutaminase-catalysed reactions.

Factor XIII: the cement of the heart after myocardial infarction?

Heart failure (HF) affects >11 million people in the USA and Europe, and ischaemic heart disease accounts for >60% of these patients.1 Despite excellent diagnostic and therapeutic standards for myocardial infarction (MI), improved early phase imaging and therapy are essential to prevent the progression of cardiac dilatation and dysfunction after acute MI. Transglutaminases (TGs) are a widely distributed group of eight isoenzymes, comprising factor XIII (FXIII) and TG type 1–type 7. They catalyse the post-translational modification of matrix proteins by the formation of isopeptide bonds, either via e-lysyl–γ-glutamyl cross-links, or through incorporation of primary amines at selected peptide-bound glutamine residues.2 Collagens, fibronectin, fibrinogen, vitronectin, osteopontin, and osteonectin are the major substrates in the cardiac matrix. Their cross-linking strengthens the matrix and prevents their degradation. Dysregulation of TG activity after MI therefore results in abnormal collagen fibrillogenesis, with resulting cardiac dilatation and dysfunction.3–5 FXIII, also called fibrin-stabilizing factor, is a pro-TG present in plasma, platelets, monocytes, and macrophages.6 Its pro-form is converted by a thrombin-dependent proteolysis into the active TG FXIIIa. The latter also catalyses the formation of covalent bonds between fibrin monomers, the final step of the blood coagulation pathway. Beside its role in this cascade, it also appears crucial for wound healing … *Corresponding author. Tel: +31 43 3882949, Fax: +31 43 3871055, Email: s.heymans{at}cardio.unimaas.nl

Type 2 Transglutaminase in Neurodegenerative Diseases: The Mitochondrial Connection

"Tissue" or type 2 Transglutaminase (TG2) is a peculiar multifunctional enzyme able to catalyse Ca(2+)-dependent post-translational modification of proteins, by establishing covalent bonds between peptide-bound glutamine residues and either lysine residues or mono- and poly-amines. In addition, it may act also as a G protein in transmembrane signalling, as a kinase, as a protein disulphide isomerase and as a cell surface adhesion mediator. The vast array of biochemical functions exerted by TG2 characterises and distinguishes it from all the other members of the transglutaminase family. Multiple lines of evidence suggest an involvement of the enzyme in neurodegenerative diseases, such as Huntington's (HD) and Parkinson (PD), and that its inhibition, either via drug treatments or genetic approaches, might be beneficial for the treatment of these syndromes. This review will exploit the recent developments in the comprehension of the role played by type 2 transglutaminase in eukaryotic cells, focusing on the role exerted by TG2 on mitochondrial physiology and on the regulation of cell death pathways at the basis of neurodegenerative diseases.

Tissue transglutaminase catalyzes the deamidation of glutamines in lens βB2- and βB3-crystallins

Tissue transglutaminase (tTG) is a Ca2+-dependent enzyme catalyzing the formation of covalent crosslinks between peptide-bound glutamine and lysine residues. Lens crystallins, including αB-crystallin and several β-crystallins, are in vitro substrates for tTG. In both human and bovine fetal lens extracts treated with commercially available guinea pig liver tTG we detected the formation of high molecular weight (HMW) aggregates containing crosslinked βB2- and βA3-crystallin. More interestingly, 2D-gel electrophoresis combined with mass spectrometry analysis revealed that glutamines present in the N-terminal arms of βB2- and βB3-crystallins deamidate readily in the presence of tTG. We found that both tTG-catalyzed crosslinking and deamidation disrupt the β-crystallin complex, suggesting that these tTG-catalyzed modifications can influence the macromolecular assembly of lens crystallins. These data together suggest that tTG can contribute to the age-related deamidation of glutamine residues of lens crystallins.

Transglutaminase Is Linked to Neurodegenerative Diseases

Transglutaminase catalyzes a covalent bond between peptide-bound glutamine residues and either lysine-bound peptide residues or mono- or polyamines. Multiple lines of evidence suggest that transglutaminase is involved in neurodegenerative diseases including Alzheimer disease, progressive supranuclear palsy, Huntington disease (HD), and Parkinson disease. In all of the neurodegenerative diseases examined to date, transglutaminase enzyme activity is upregulated in selectively vulnerable brain regions, transglutaminase proteins are associated with inclusion bodies characteristic of the diseases, and prominent proteins in the inclusion bodies are modified by transglutaminase enzymes. These prominent proteins in the inclusion bodies, including tau, alpha-synuclein, and huntingtin protein, are modified by transglutaminase in vitro and alpha-synuclein and huntingtin protein are modified in cells in culture. Similar changes in transglutaminase and transglutaminase-modified proteins are replicated in transgenic mouse models of the neurodegenerative diseases, including Huntington disease and progressive supranuclear palsy. Lastly, inhibition of transglutaminase either via drug treatments or molecular approaches is beneficial for the treatment of HD transgenic mice but has yet to be explored for the other neurodegenerative diseases. Further research is needed to determine the specific role(s) that transglutaminase plays in the pathophysiology of neurodegenerative diseases with possible implications for transglutaminase as a therapeutic target.

Acyl transfer from carboxylate, carbonate, and thiocarbonate esters to enzymatic and nonenzymatic thiolates

Transglutaminases (EC 2.3.2.13) (TGases) catalyze calcium-dependent acyl transfer reactions between peptide-bound glutamine residues as acyl donors and peptide-bound lysine residues as acyl acceptors, resulting in the formation of intermolecular ε-(γ-glutamyl)lysine crosslinks. The mechanistic details of its "ping-pong" transamidation reaction remain unknown. In particular, few studies have been published probing the nucleophilicity of TGase using acyl-donor substrates of varied electrophilicity. Herein we report the synthesis of activated esters of carbonates, carbamates, and thiocarbonates and their reactions with simple thiols, as a nonenzymatic point of reference, and with the catalytic cysteine residue of guinea pig liver TGase. Our kinetic results show that the simple substitution of a side chain methylene unit by oxygen or sulphur had a surprising effect on both substrate affinity and acylation reactivity. Furthermore, they provide unexpected insight into the importance of a side chain heteroatom for conferring affinity for tissue TGase as well as revealing an interesting class of irreversible inhibitors.Key words: enzyme kinetics, enzyme inhibition, transglutaminase, acyl-transfer reactions, carbamate, thiocarbonate, carbonate.

Cross-Linking Transglutaminases with G Protein–Coupled Receptor Signaling

Transglutaminases are a family of calcium- and thiol-dependent acyl transferases that catalyze the formation of an amide bond between the gamma-carboxamide groups of peptide-bound glutamine residues and the primary amino groups in various compounds, including the epsilon-amino group of lysines in certain proteins. As a result, these enzymes effect posttranslational modification of proteins by amine incorporation, or stabilization of protein assemblies by their cross-linking; such actions profoundly influence critical biological processes such as blood clotting and protection from infection and dehydration by establishing the barrier function of skin. In addition, transglutaminases have other more diverse actions, including involvement in signaling by the superfamily of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) in one of three ways: (i) through actions as guanosine triphosphate-binding proteins that activate intracellular effectors, such as phospholipase C; (ii) by cross-linking GPCR monomers to enhance signaling as a result of covalent dimer formation; or (iii) by interacting with an apparent growth inhibitory orphan GPCR, GPR56, to limit metastatic spread of melanoma cells. The implications of these receptor-coupled actions of transglutaminases are discussed.

Tissue transglutaminase: from biological glue to cell survival cues

Tissue transglutaminase (TG2, EC 2.3.2.13) is a ubiquitous enzyme that catalyzes Ca2+-dependent post-translational modification of proteins by inserting highly stable (epsilon-[gamma-glutamyl] lysine) isopeptide bonds or by conjugating polyamines at selected peptide-bound glutamine residues. The TG2-catalyzed cross-linked products (generally high molecular mass scaffold of proteins) are of great physiological significance; they are highly stable and resistant to mechanical, chemical and proteolytic degradation. The accumulation of isopeptide bonds can be observed in skin, hair and during blood clotting and wound healing. In addition to transamidation activity, TG2 also exhibits GTPase activity and in response to certain agonist hormones can serve as a signal transducing G protein. Although predominantly a cytosolic protein, TG2 can translocate to the nucleus with the help of importin alpha-3 protein or to the membranes in association with integrins. Moreover, TG2 can also be secreted outside the cell (by yet unknown mechanism) where it crosslinks proteins of the extracellular matrix (ECM) and promotes cell adhesion and spreading. Another important property of TG2 is that it has high binding-affinity for the ECM component protein, fibronectin and thus can promote interaction between cell surface integrin with fibronectin. In this review, we discuss the implications of increased TG2 expression in drug-resistant and metastatic cancer cells and that how TG2 expression can contribute in the development of these phenotypes.

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

Previous reports have suggested that protein disulfide isomerases (PDIs) have transglutaminase (TGase) activity [1]. The structural basis of this reaction has not been revealed. We demonstrate here that Caenorhabditis elegans PDI-3 can function as a Ca 2+-dependent TGase in assays based on modification of protein- and peptide-bound glutamine residues. By site-directed mutagenesis the second cysteine residue of the –CysGlyHisCys– motif in the thioredoxin domain of the enzyme protein was found to be the active site of the transamidation reaction and chemical modification of histidine in their motif blocked TGase activity.

Transglutaminases: nature's biological glues.

Transglutaminases (Tgases) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of isopeptide bonds. This occurs either through protein cross-linking via epsilon-(gamma-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation, and their accumulation is found in a number of tissues and processes where such properties are important, including skin, hair, blood clotting and wound healing. However, deregulation of enzyme activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin. In the present review we detail the structural and regulatory features important in mammalian Tgases, with particular focus on the ubiquitous type 2 tissue enzyme. Physiological roles and substrates are discussed with a view to increasing and understanding the pathogenesis of the diseases associated with transglutaminases. Moreover the ability of these enzymes to modify proteins and act as biological glues has not gone unnoticed by the commercial sector. As a consequence, we have included some of the present and future biotechnological applications of this increasingly important group of enzymes.

Histone cross-linking by transglutaminase

Transglutaminases irreversibly catalyze covalent cross-linking of proteins by forming isopeptide bonds between peptide-bound glutamine and lysine residues. Among several transglutaminases, tissue-type transglutaminase (tTGase) is most ubiquitously found in every type of cells and tissues in animals, but its natural substrate has yet to be identified. In an attempt to identify the natural substrate for tTGase, we examined in vitro if core histones were subject to cross-linking by tTGase. We found core histone subunits, H2A and H2B, were specifically cross-linked by tTGase. The cross-linking was between either one or both glutamines at C-terminal end of H2A (-VTIA Q 104 GGVLPNT Q 112 SVLLPKKTESSKSK—C ′ end) and the first and/or third lysine from C-terminal end of H2B (-AVESEG K 116 AVTKYTSS K 125 —C ′ end). The cross-linking occurred only when these subunits were released from nucleosome but not when these were organized in nucleosome. Most interestingly, in chicken erythrocyte the cross-linked H2A–H2B was present in a significant amount. From these results, it can be proposed that tTGase-mediated cross-linking is an another form of core histone modification and it may play a role of chromatin condensation during erythrocyte differentiation.

Transglutaminase-3, an esophageal cancer-related gene.

Transglutaminase-3 (TGase-3) is an enzyme with the ability to catalyze the irreversible cross-linking of peptide-bound glutamine residues either with peptide-bound lysines or with primary amines. It has been implicated in the formation and assembly of the cornified cell envelope of the epidermis, hair follicle and perhaps other stratified squamous epithelia. We show here the involvement of TGase-3 in human esophageal cancer. In an initial study, mRNA differential display was performed with 3 pairs of esophageal cancer tissues and matched normal adjacent mucosa by a 10-mer arbitrary primer and mixed anchored primers (GT15N, N = A, C and G). Four differentially expressed cDNA bands were consistently observed in all 3 normal tissues but barely detected in their tumor counterparts. One of them was identified to be the 3; end of TGase-3. Northern blot and dot blot analyses of 14 samples confirmed the down-regulation of TGase-3 in malignant tissues compared with normal epithelia. RT-PCR revealed that TGase-3 expression was lost in 3 esophageal carcinoma cell lines and decreased in 35/38 tumors compared with adjacent normal mucosa. Taken together, 49/52 (94.2%) esophageal tumors presented down-regulation of the gene. Our data suggest that alteration of TGase-3 expression is a common event in the development of human esophageal cancer.

Expression of tissue-type transglutaminase correlates positively with metastatic properties of human melanoma cell lines.

In this study the relationship between tissue-type transglutaminase (TGase2) activity and the propensity to metastasize was investigated in human melanoma cell lines with different metastatic behavior. TGase2 catalyzes an acyl-transfer reaction between peptide-bound glutamine residues and primary amines, including the epsilon-amino group of lysine residues. Northern-blot analysis demonstrated that TGase2 RNA-expression (3.7 kb) was elevated in highly metastatic cell lines (MV3 and BLM) as compared to weakly metastatic ones (IF6 and 530). Immunoprecipitation and enzyme assays of TGase2 showed that the differential expression at the mRNA level was also reflected at the protein level. These findings reveal a positive relation between the expression of TGase2 and the metastatic properties of the human melanoma cell lines.