Preparation and mechanical properties of edible pectin-soy flour films obtained in the absence or presence of transglutaminase

Whole soy flour and apple pectin were used as raw materials for producing hydrocolloid edible films. The best ratio between the two components (2:1 mg cm(-2), pectin-soy flour) was determined in order to obtain films which could be perfectly handled for their consistence. Films were also prepared in the presence of transglutaminase, an enzyme able to produce isopeptide bonds among the soy polypeptide chains. The latter films showed a smoother surface and higher homogeneity, as demonstrated by microstructural analyses, whereas studies on the mechanical properties indicated that transglutaminase increased their strength and reduced their flexibility. Our results suggest a possible use of the transglutaminase polymerized pectin-soy protein films as edible food or drug coatings.     

Fennel waste-based films suitable for protecting cultivations

Biodegradable, flexible, and moisture-resistant films were obtained by recycling fennel waste and adding to fennel homogenates the bean protein phaseolin that was modified or not modified by the enzyme transglutaminase. All films were analyzed for their morphology, mechanical properties, water vapor permeability, and susceptibility to biodegradation under soil-like conditions. Our experiments showed that transglutaminase treatment of the phaseolin-containing fennel waste homogenates allowed us to obtain films comparable in their mechanical properties and water vapor permeability to the commercial films Ecoflex and Mater-Bi. Furthermore, biodegradability tests demonstrated that the presence of the enzyme in the film-casting sample significantly influences the integrity of such a product that lasts longer than films obtained either with fennel waste alone or with a mixture of fennel waste and phaseolin. These findings indicate the fennel-phaseolin film prepared in the presence of transglutaminase to be a promising candidate for a new environmentally friendly mulching bioplastic.     

Putrescine–polysaccharide conjugates as transglutaminase substrates and their possible use in producing crosslinked films

Putrescine (1,4-diaminobutane) was covalently linked to alginate and low-methoxyl pectin to synthesize new aminated polysaccharides. Both putrescine–pectin and –alginate conjugates, although the latter at higher concentrations, were found to be able to act as effective acyl acceptor transglutaminase substrates in vitro using both dimethylated casein and soy flour proteins as acyl donors. Monodansylcadaverine, a well known acyl acceptor transglutaminase substrate, dose-dependently counteracted the covalent binding of the aminated polysaccharides to the proteins. Putrescine–pectin conjugate was also tested to prepare, in combination with soy flour proteins, edible films in the presence of purified microbial transglutaminase. Characterization of the enzymatically crosslinked films showed a significant decreased water vapor permeability, with respect to the ones obtained with non-aminated pectin in the presence of transglutaminase, as well as improved mechanical properties, such as high extensibility. Possible biotechnological applications of hydrocolloid films containing putrescine–polysaccharide derivatives enzymatically crosslinked to proteins were suggested.     

Endogenous substrates for epidermal transglutaminase.

Potential in vivo substrates for epidermal transglutaminase have been isolated and partially characterized in human stratum corneum and new born rat epidermis. [14C]Putrescine and dansylcadaverine were incorporated into epidermal proteins in vitro. Two high molecular weight proteins incorporated the labels in both the rat ahd human homogenates. One of the proteins was too large to enter a 4% sodium dodecyl sulfate-polyacrylamide spacer gel; the other was seen at the interface between the spacer gel and a 10% sodium dodecyl sulphate-polyacrylamide running gel. These proteins were present in a buffer extract, sodium dodecyl sulphate-dithiothreitol extract and NaOH extract. The labels were also incorporated into protein in the insoluble pellet remaining after the afore-mentioned extractions. The incorporation of putrescine and dansylcadaverine was time dependent, and was inhibited by known inhibitors of epidermal transglutaminase. The two high molecular weight proteins had similar amino acid composition, characterized by high glycine, glutamic acid, serine and aspartic acid. The amino acid composition was similar to, although not identical with, the amino acid composition of alpha-keratin proteins. Epidermal homogenates incubated in the presence of transglutaminase showed progressive insolubilization of the protein. This cross-linking was inhibited by putrescine. [14C]Glycine, [14C]histidine and [4C]proline were incorporated into epidermal proteins in newborn rats in vivo. The glycine-labelled protein became progressively more insoluble when incubated in vitro in the presence of transglutaminase. In vitro incubation with transglutaminase had no effect on the histidine-and proline-labelled proteins.     

Regulation of transglutaminase type II by transforming growth factor-beta 1 in normal and transformed human epidermal keratinocytes

This study examines the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of Type I and II transglutaminase in normal human epidermal keratinocytes (NHEK cells). Treatment of undifferentiated NHEK cells with 100 pM TGF-beta 1 caused a 10- to 15-fold increase in the activity of a soluble transglutaminase. Based on its cellular distribution and immunoreactivity this transglutaminase was identified as Type II (tissue) transglutaminase. TGF-beta 1 did not enhance the levels of the membrane-bound Type I (epidermal) transglutaminase activity which is induced during squamous cell differentiation and did not increase Type II transglutaminase activity in differentiated NHEK cells. Several SV40 large T antigen-immortalized NHEK cell lines also exhibited a dramatic increase in transglutaminase Type II activity after TGF-beta 1 treatment; however, TGF-beta 1 did not induce any significant change in transglutaminase activity in the carcinoma-derived cell lines SCC-13, SCC-15, and SQCC/Y1. Half-maximal stimulation of transglutaminase Type II activity in NHEK cells occurred at a dose of 15 pM TGF-beta 1. TGF-beta 2 was about equally effective. This enhancement in transglutaminase activity was related to an increase in the amount of transglutaminase Type II protein as indicated by immunoblot analysis. Northern blot analyses using a specific cDNA probe for Type II transglutaminase showed that exposure of NHEK cells to TGF-beta 1 caused a marked increase in the mRNA levels of this enzyme which could be observed as early as 4 h after the addition of TGF-beta 1. Maximal induction of transglutaminase Type II mRNA occurred between 18 and 24 h. The increase in Type II transglutaminase mRNA levels was blocked by the presence of cycloheximide, suggesting that this increase in mRNA by TGF-beta 1 is dependent on protein synthesis.     

Expression of tissue transglutaminase in skeletal tissues correlates with events of terminal differentiation of chondrocytes

Calcifying cartilages show a restricted expression of tissue transglutaminase. Immunostaining of newborn rat paw bones reveals expression only in the epiphyseal growth plate. Tissue transglutaminase appears first intracellularly in the proliferation/maturation zone and remains until calcification of the tissue in the lower hypertrophic zone. Externalization occurs before mineralization. Subsequently, the enzyme is present in the interterritorial matrix during provisional calcification and in the calcified cartilage cores of bone trabeculae. In trachea, mineralization occurring with maturation in the center of the cartilage is accompanied by expression of tissue transglutaminase at the border of the hydroxyapatite deposits. Transglutaminase activity also shows a restricted distribution in cartilage, similar to the one observed for tissue transglutaminase protein. Analysis of tissue homogenates showed that the enzyme is present in growth plate cartilage, but not in articular cartilage, and recognizes a limited set of substrate proteins. Osteonectin is coexpressed with tissue transglutaminase both in the growth plate and in calcifying tracheal cartilage and is a specific substrate for tissue transglutaminase in vitro. Tissue transglutaminase expression in skeletal tissues is strictly regulated, correlates with chondrocyte differentiation, precedes cartilage calcification, and could lead to cross-linking of the mineralizing matrix.     

Retinol-induced morphological changes of cultured bovine endothelial cells are accompanied by a marked increase in transglutaminase

Retinol, a morphogen, has been shown to induce morphological changes in vascular endothelial cells, accompanied by an acute and specific accumulation of an 80-kDa protein; purification and characterization of this retinol-induced protein (RIP) have revealed that it is a transglutaminase. Endothelial cells from bovine carotid artery were cultured, treated with retinol, and examined for changes in morphology and protein profiles. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of extracts prepared from retinol-treated cells which had undergone a remarkable change in shape (from a cobblestone-like to a spindle-like shape) indicated that the retinol-induced morphological change is accompanied by a marked increase of an 80-kDa protein. Similar changes were also induced by retinoic acid. The 80-kDa RIP was purified by anion exchange and hydroxyapatite column chromatography. Amino acid sequencing of tryptic fragments of the purified RIP revealed a high degree of homology between the sequence of bovine RIP and that of guinea pig liver transglutaminase, suggesting that RIP is a transglutaminase. This was confirmed by activity measurements; RIP exhibited transglutaminase activity, and an antiserum against RIP immunoprecipitated the activity. These results suggest that transglutaminase plays important roles in the maintenance of morphology and the control of endothelial cell functions.     

Induction of cellular transglutaminase biosynthesis by sodium butyrate

Cellular transglutaminase activity was induced in simian virus-transformed human embryonic lung fibroblasts (WI-38 VA13A) by sodium butyrate. The level of enzyme activity approached a maximum by 6 days; 9–11-fold higher in the presence of sodium butyrate (1 mM) than in its absence. The observed increases in cellular transglutaminase activity could be entirely accounted for by equivalent increases in the levels of enzyme protein measured by inhibition enzyme-linked immunosorbent assay. Sodium butyrate also increased the rate of enzyme synthesis, but had no effect on the rate of cellular transglutaminase degradation. The increase in the rate of enzyme synthesis was matched by an increased level of translatable transglutaminase mRNA as measured in a cell-free translation system. Our results suggest that sodium butyrate regulates cellular transglutaminase at the pretranslational level.     

Gelation of Casein and Soybean Globulins by Transglutaminase

Guinea pig liver transglutaminase is a Ca²⁺ dependent enzyme which catalyzes the formation of inter- and intramolecular ε-(γ-glutamyl)lysyl cross-links between protein molecules. We have found that solutions of several proteins (α_s1-casein, and soybean 11S and 7S globulins) were gelatinized firmly by transglutaminase. The gel formation depended on the protein concentration. In the case of α_s1-casein, a reaction mixture containing below 2% was incapable of gelation. However, above 3%, a firm gel was formed by transglutaminase. As to soybean 11S and 7S globulins, reaction mixtures containing below 5% did not form gels, while, above 8%, firm gels were formed. The protein solutions in the presence of EDTA, an inhibitor of transglutaminase, were not gelatinized on treatment with transglutaminase. Thus, transglutaminase and a higher concentration of a substrate protein are indispensable for firm gel formation. It is supposed that the protein gels are formed through covalent bonds with transglutaminase.     

Ligand-induced conformational changes in tissue transglutaminase: Monte Carlo analysis of small-angle scattering data

Small-angle neutron and x-ray scattering experiments have been performed on type 2 tissular transglutaminase to characterize the conformational changes that bring about Ca(2+) activation and guanosine triphosphate (GTP) inhibition. The native and a proteolyzed form of the enzyme, in the presence and in the absence of the two effectors, were considered. To describe the shape of transglutaminase in the different conformations, a Monte Carlo method for calculating small-angle neutron scattering profiles was developed by taking into account the computer-designed structure of the native transglutaminase, the results of the Guinier analysis, and the essential role played by the solvent-exposed peptide loop for the conformational changes of the protein after activation. Although the range of the neutron scattering data is rather limited, by using the Monte Carlo analysis, and because the structure of the native protein is available, the distribution of the protein conformations after ligand interaction was obtained. Calcium activation promotes a rotation of the C-terminal with respect to the N-terminal domain around the solvent-exposed peptide loop that connects the two regions. The psi angle between the longest axes of the two pairs of domains is found to be above 50 degrees, larger than the psi value of 35 degrees calculated for the native transglutaminase. On the other hand, the addition of GTP makes possible conformations characterized by psi angles lower than 34 degrees. These results are in good agreement with the proposed enzyme activity regulation: in the presence of GTP, the catalytic site is shielded by the more compact protein structure, while the conformational changes induced by Ca(2+) make the active site accessible to the substrate.     

Identification of a guanosine triphosphate-binding site on guinea pig liver transglutaminase. Role of GTP and calcium ions in modulating activity

Guanosine 5'-triphosphate (GTP) was found to inhibit guinea pig liver transglutaminase activity as measured by [3H]putrescine incorporation into casein. GDP and GTP-gamma-S also inhibited enzyme activity (GTP-gamma-S greater than GTP greater than GDP). Kinetic studies showed that GTP acted as a reversible, noncompetitive inhibitor and that CaCl2 partially reversed GTP inhibition. GTP also inhibited rat liver and adult bovine aortic endothelial cell transglutaminase, but did not inhibit Factor XIIIa activity. Guanosine monophosphate (GMP), cyclic GMP, and polyguanylic acid did not inhibit enzyme activity. Guinea pig liver transglutaminase adsorbed well to GTP-agarose affinity columns, but not to CTP-agarose columns, and the binding was inhibited by the presence of calcium ions. Specific binding of GTP to transglutaminase was demonstrated by photoaffinity labeling with 8-azidoguanosine 5'-[gamma-32P] triphosphate, which was inhibited by the presence of GTP or CaCl2. GTP inhibited trypsin proteolysis of guinea pig liver transglutaminase without affecting the trypsin proteolysis of chromogenic substrates. Proteolytic protection was reversed by the addition of calcium. This study demonstrates that GTP binds to transglutaminase and that both GTP and calcium ions function in concert to regulate transglutaminase structure and function.     

Transglutaminase 2 interaction with small heat shock proteins mediate cell survival upon excitotoxic stress

Transglutaminase 2 has been postulated to be involved in the pathogenesis of central nervous system neurodegenerative disorders. However, its role in neuronal cell death remains to be elucidated. Excitotoxicity is a common event underlying neurodegeneration. We aimed to evaluate the protein targets for transglutaminase 2 in cell response to NMDA-induced excitotoxic stress, using SH-SY5Y neuroblastoma cells which express high tranglutaminase 2 levels upon retinoic acid-driven differentiation toward neurons. NMDA-evoked calcium increase led to transglutaminase 2 activation that mediated cell survival, as at first suggested by the exacerbation of NMDA toxicity in the presence of R283, a synthetic competitive inhibitor of transglutaminase active site. Assays of R283-mediated transglutaminase inhibition showed the involvement of enzyme activity in NMDA-induced reduction in protein basal levels of pro-apoptotic caspase-3 and the stress protein Hsp20. However, this occurred in a way different from protein cross-linking, given that macromolecular assemblies were not observed in our experimental conditions for both proteins. Co-immunoprecipitation experiments provided evidence for the interaction, in basal conditions, between transglutaminase 2 and Hsp20, as well as between Hsp20 and Hsp27, a major anti-apoptotic protein promoting caspase-3 inactivation and degradation. NMDA treatment disrupted both these interactions that were restored upon transglutaminase 2 inhibition with R283. These results suggest that transglutaminase 2 might be protective against NMDA-evoked excitotoxic insult in neuronal-like SH-SY5Y cells in a way, independent from transamidation that likely involves its interaction with the complex Hsp20/Hsp27 playing a pro-survival role.     

Consequences of terbium (111) binding on the conformation and enzymatic activity of Guinea pig liver transglutaminase

Calcium ions are crucial for expression of transglutaminase activity. Although lanthanides have been reported to substitute for calcium in a variety of protein functions, they did not replace the calcium requirement during transglutaminase activity measurements. Furthermore, lanthanides strongly inhibited purified liver transglutaminase activity using either casein or fibrinogen as substrates. Terbium (III) inhibition of transglutaminase-catalyzed putrescine incorporation into casein was not reversed by the presence of 10–200 fold molar excess of calcium ions (Ki for Tb(III)=60 µM). Conformational changes in purified liver transglutaminase upon Tb(III) binding were evident from a biphasic effect of Tb(III) on transglutaminase binding to fibrin. Low concentrations of Tb(III) (1 µM to 10 µM inhibited the binding of transglutaminase to fibrin, whereas higher concentrations (20 µM to 100 µM promoted binding. Conformational changes in purified liver transglutaminase consequent to Tb(III) binding were also demonstrated by fluorescence spectroscopy due to Forster energy transfer. Fluorescence emission was stable to the presence of 200 mM NaCl and 100 mM CaCl₂ only partially quenched emission. Purified liver transglutaminase strongly bound to Tb(III)-Chelating Sepharose beads and binding could not be disrupted by 100 mM CaCl₂ solution. Our data suggest that Tb(III)-induced conformational changes in transglutaminase are responsible for the observed effects on enzyme structure and function. The potential applications of Tb(III)-transglutaminase interactions in elucidating the structure-function relationships of liver transglutaminase are discussed.     

Regulation of transglutaminase activity in Chinese hamster ovary cells

We have investigated the regulation of transglutaminase activity (epsilon-(gamma-glutamyl)lysine crosslinking enzyme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20--70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.     

Characterization of purified rat testicular transglutaminase and age-dependent changes of the enzyme activities

The Ca2+-dependent tissue transglutaminase is widely distributed in various tissues and has been reported to participate in many cellular growth and differentiation processes. In the past decade, tissue transglutaminase is also identified as a G protein, G(alphah), for intercellular signaling. To further characterize testicular transglutaminase, the rat testicular transglutaminase was purified by ammonium sulfate precipitation, DEAE ion-exchange, heparin-agarose, and GTP-agarose affinity chromatographies. This purification protocol resulted in a 8400-fold enrichment of the enzyme with a reproducible 15% yield. The purified enzyme showed as a single band of 78kDa on SDS-polyacrylamide gel. Western blot analysis using anti-liver tissue transglutaminase monoclonal antibody also recognized the enzyme, indicating it is a t-TGase in nature. The Km values of purified testicular transglutaminase for putrescine and N,N-dimethylcasein were determined to be 35 and 17 microM, respectively. Its transglutaminase cross-linking activity was strongly inhibited by EGTA, GTP, polyamines, and cystamine, as well as moderately by ATP and NaCl. The enzyme exhibited a magnesium-dependent GTP-hydrolyzing capacity, but its GTP-binding activity did not require magnesium. Furthermore, the enzyme activity was found to be closely related with the first wave of spermatogenesis. Thus, testicular transglutaminase is speculated to participate in the event of spermatogenesis. In conclusion, the purified testicular transglutaminase displays property of either the tissue-type transglutaminase, or the GTP-binding and hydrolyzing characteristics. The activity of testicular transglutaminase is age-dependent, greatly stimulated during the first wave of spermatogenesis.     

Critical role of transglutaminase and other stress proteins during neurodegenerative processes

Proteolytic stress, resulting from the intracellular accumulation of misfolded or aggregated proteins, which exceed the capacity of the ubiquitin–proteasome system to degrade them, plays a relevant role in neurodegenerative disorders, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s chorea. Most of toxic protein aggregates are characterised by the presence of isopeptide bonds (cross-links) catalysed by transglutaminase activity; further, several disease-specific proteins—tau, amyloid-beta, alpha-synuclein, huntingtin—are in vitro and/or in vivo substrates of transglutaminase 2. These findings suggest an important role for transglutaminase 2-mediated cross-linking reactions in neurodegeneration. Therefore, the use of transglutaminase activity inhibitors could ameliorate neuronal cell death. New therapeutic perspectives also arise from the possibility to prevent or reduce protein aggregation by enhancing the activation of heat shock proteins, which have been shown to be potent suppressors of neurodegeneration in cell cultures/animal models. Interestingly, some heat shock proteins have been shown to be in vitro or in vivo cross-linked by transglutaminase 2. These observations seem to suggest that transglutaminase activity could be involved in the stabilization of intracellular protein aggregates by interfering with proteasomal degradation of misfolded proteins. Further studies are needed to validate leading hypotheses and to open new prospects for developing therapeutic tools.     

The length of polyglutamine tract, its level of expression, the rate of degradation, and the transglutaminase activity influence the formation of intracellular aggregates.

A common feature of CAG-expansion neurodegenerative diseases is the presence of intranuclear aggregates in neuronal cells. We have used a synthetic fusion protein containing at the NH2 terminus the influenza hemoagglutinin epitope (HA), a polyglutamine stretch (polyQ) of various size (17, 36, 43 CAG) and a COOH tail encoding the green fluorescent protein (GFP). The fusion proteins were expressed in COS-7 and neuroblastoma SK-N-BE cells. We found that the formation of aggregates largely depends on the length of polyglutamine tracts and on the levels of expression of the fusion protein. Moreover, transglutaminase overexpression caused an increase of insoluble aggregates only in cells expressing the mutant expanded protein. Conversely, treatment of cells with cystamine, a transglutaminase inhibitor, reduced the percentage of aggregates. We found also that the inhibition of the proteasome ubiquitin-dependent degradation increased the formation of intranuclear aggregates. These data suggest that length of polyglutamine tract, its expression, unbalance between cellular transglutaminase activity, and the ubiquitin-degradation pathway are key factors in the formation of intranuclear aggregates.     

The binding sites on fibrin(ogen) for guinea pig liver transglutaminase are similar to those of blood coagulation factor XIII. Characterization of the binding of liver transglutaminase to fibrin

The present study represents detailed investigations into the nature of interactions between an intracellular "tissue" transglutaminase and a plasma protein, fibrinogen. We demonstrate a specific, saturable, and reversible binding of transglutaminase to fibrin(ogen). The binding was time- and temperature-dependent, was independent of divalent metal ions, did not require the release of either fibrinopeptide A or B, and was partially inhibited by the presence of sodium chloride or plasma proteins, properties similar to Factor XIII binding to fibrin(ogen). Both Factor XIII and liver transglutaminase also shared similar binding sites on fibrinogen, the A alpha- and the B beta-chains. The binding characteristics of liver transglutaminase were thus similar to Factor XIII binding to fibrin, but there were also important differences. Scatchard analyses of the binding data indicated that the affinity of liver transglutaminase (Kd = 4.17 x 10(-7) M) was at least 40-fold weaker compared with the affinity of Factor XIII to fibrinogen. Consequently, a 20-fold molar excess of Factor XIII a-chains specifically and completely inhibited the binding of liver transglutaminase to des-A-fibrinogen. The association between liver transglutaminase and fibrin(ogen) was also critically controlled by the conformational states of the two proteins. Substances capable of altering the conformation of either transglutaminase (such as guanosine 5'-triphosphate) or of fibrinogen (such as the tetrapeptide Gly-Pro-Arg-Pro and Fragment D) disrupted binding. Excess CaCl2 was able to counteract the effects of guanosine 5'-triphosphate on transglutaminase binding to fibrin. In contrast, Factor XIII binding to fibrin was unaffected by either guanosine 5'-triphosphate, CaCl2, or Gly-Pro-Arg-Pro, suggesting a more stable association between the two proteins. The physiologic implications of transglutaminase-fibrin(ogen) interactions are discussed.     

Transglutaminase-catalyzed modifications of SV-IV, a major protein secreted from the rat seminal vesicle epithelium

One of the major proteins secreted from the rat seminal vesicle epithelium, namely SV-IV, was shown to act in vitro as acyl donor and acceptor substrate for transglutaminase from both guinea pig liver and rat anterior prostate secretory fluid. Electrophoretic and chromatographic experiments indicated that the enzyme catalyzed the formation of multiple modified forms of SV-IV. In the absence of small Mr amines, transglutaminase was able to produce at least six different molecular forms of the protein, half of which possessed an Mr higher than that of native SV-IV. These findings suggested that a variable number of intermolecular, and perhaps intramolecular, crosslinks were formed between one or both glutamine residues and one or more lysine residues occurring in the SV-IV polypeptide chain. In addition, at least three modified forms of the protein were produced by transglutaminase in the presence of high concentrations of spermidine, thus indicating the formation of different (gamma-glutamyl)polyamine derivatives of SV-IV. Rabbit uteroglobin and rat anterior prostate secretory protein(s) were also shown to be able to covalently bind spermidine in the presence of the enzyme. The possible biological significance of transglutaminase-mediated modifications of SV-IV, as well as of other proteins occurring in the mammal seminal fluid, are discussed.     

Purification and partial characterization of the plasma clotting protein from the pink shrimp Farfantepenaeus paulensis

A clotting protein (CP) was purified from the plasma of the pink shrimp Farfantepenaeus paulensis by sequential anion-exchange chromatography. The shrimp CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca2+, suggesting that the clotting reaction is catalyzed by a Ca2+-dependent transglutaminase present in shrimp hemocytes. Dansylcadaverine was incorporated into the shrimp CP in the presence of endogenous transglutaminase (hemocyte lysate), confirming that the shrimp purified CP is the substrate for the transglutaminase enzyme. The molecular mass of the CP was determined by gel filtration to be 341 kDa and 170 kDa by SDS-PAGE under reducing conditions. These results suggest that the shrimp CP consists of two identical subunits, covalently linked by disulphide bonds. The amino acid sequence at the N-terminus was 100% identical to that of the penaeids Litopenaeus vannamei and Penaeus monodon and 66% to 80% identical to the CPs of other decapods. This is the first report of a CP characterization in an Atlantic penaeid species. Further studies, including a molecular cloning approach would enable to detect which tissues express the gene of the clotting protein. It would be also useful to understand the mechanism by which the coagulation time is delayed in shrimps under stress conditions.