Transglutaminase-mediated macromolecular assembly: production of conjugates for food and pharmaceutical applications.

Various strategies have been explored in the last 20 years to modify the functional properties of proteins and, among these, protein/polymer conjugation resulted one of the most successful approaches. Thus, the surface modification of polypeptides of potential industrial interest by covalent attachment of different macromolecules is nowadays regarded as an extremely valuable technique to manipulate protein activities. Protein derivatives with a number of either natural or synthetic polymers, like different polysaccharides or polyethylene glycol, have been obtained by both chemical and enzymatic treatments, and in this context, the crosslinking enzyme transglutaminase is attracting an increasing attention as a simple and safe means for protein processing in vitro. In this short review, we summarized the most significant experimental findings demonstrating that a microbial form of the enzyme is an effective tool to obtain several biopolymer-based conjugates potentially useful for both food and pharmaceutical applications.

Swelling, mechanical, and barrier properties of albedo-based films prepared in the presence of phaseolin cross-linked or not by transglutaminase.

Edible films were obtained from Citrus paradisi grapefruit albedo homogenates and bean protein phaseolin modified or not by the enzyme transglutaminase. Swelling capability, barrier performance to water vapor, oxygen and carbon dioxide, and mechanical properties of such films were investigated. The addition of the protein, mostly in the presence of transglutaminase, provide films less swellable at pH values above 5 compared to films made by albedo homogenates only, whereas the action of the enzyme clearly improves mechanical properties producing more stretchable and elastic films. Moreover, transglutaminase-mediated cross-linking of phaseolin gives rise to films less permeable to carbon dioxide and able to offer a high barrier to water vapor. These findings suggest that albedo-phaseolin film prepared in the presence of transglutaminase can be a promising candidate to be used as food edible wrap.

Recombinant human tissue transglutaminase produced into tobacco suspension cell cultures is active and recognizes autoantibodies in the serum of coeliac patients.

Human tissue transglutaminase (htTG) is one of the most important member within the transglutaminase family, enzymes that for their capacity of catalyzing post-translational modifications of proteins and peptides, rise an high interest for industrial applications. More recently, for its implication as the major autoantigen in the coeliac disease, availability of human tissue transglutaminase as recombinant form is required for accurate diagnostic tests. The aim of this study was to find an alternative and inexpensive source to produce human tissue transglutaminase. To date, plant systems are proposed as heterologous hosts to produce recombinant proteins for use in disease diagnosis and therapy. Here, we describe the stable expression of human tissue transglutaminase into Nicotiana tabacum cultured cells (cultivar Bright Yellow 2 (BY-2)). The recombinant enzyme was successfully expressed in different plant cell compartments and both apoplast (apo) and chloroplast (chl) purified proteins were shown to be catalytically active and able to bind GTP, a property possessed by the natural counterpart. Importantly, plant produced human tissue transglutaminase recognized autoantibodies in the serum of coeliac patients, suggesting possible applications in the diagnosis of coeliac disease.

Impact of dehulling on the physico-chemical properties and in vitro protein digestion of common beans (Phaseolus vulgaris L.).

The objective of this study was to understand the effect of dehulling on the microstructural, physico-chemical characteristics, and in vitro protein digestion of common bean flours with particular regard to differences between adults and infants. The microstructure of flour samples from undehulled (WB) and manually dehulled (SB) beans, observed through scanning electron microscopy, showed that WB starch granules appeared to be surrounded by an integral matrix, while the SB starch granule structure was still visible although covered by protein clusters. The starch granules were oval and spherical, with heterogeneous sizes ranging from 19 to 30 µm in diameter. Particle size analysis determined with a laser diffraction particle size analyzer showed similar bimodal particle size distributions of small (1-25 µm) and large (>100 µm) granules, though the particle size of WB was obviously higher than SB. Color and other physico-chemical analyses showed that dehulling had significant (P < 0.05) influence on all investigated characteristics. The in vitro gastric and duodenal digestion experiments carried out under physiological conditions showed that the SB samples are more likely to be digested by infants. From our results, it is possible to conclude that the dehulling process improves bean flour protein digestion which could be utilized in various food applications.

Substance P inactivation by transglutaminase in vitro.

Gamma(glutamyl5)spermine derivative of substance P (Spm-SP) was synthesized in vitro in the presence of purified guinea pig liver transglutaminase and Ca2+. The spermine adduct of the neuropeptide was purified by HPLC on a reversed-phase column and characterized by fast atom bombardment mass spectrometry. The biological activities of Spm-SP were tested by assaying, in comparison with substance P, its ability to induce both the contractions of smooth muscle in vitro and the edema formation in vivo. Spm-SP was shown not to elicit contractile responses in the isolated rat stomach strip and duodenum and not to antagonize the spasmogenic effect evoked by the native neuropeptide. Furthermore, Spm-SP was unable, when administered into rats by plantar injection, either to provoke an acute inflammatory response in the hind limb or to antagonize the edema formation induced by a concurrent administration of substance P. These results indicate that the introduction of a large size hydrophilic moiety at the glutamine5 level negatively affects the ability of the neuropeptide to bind to its receptor(s), thus supporting the view that the hydrophobic middle portion of substance P plays a key role in receptor recognition.

Transglutaminase-synthesized gamma-(glutamyl5) spermidine derivative of substance P is a selective tool for neurokinin-2 receptors characterization.

The ability of transglutaminase-synthesized 1,3-diaminopropane, spermidine (Spd), spermine (Spm), and monodansylcadaverine gamma-(glutamyl5)derivatives of substance P (SP) to produce bronchoconstriction was investigated. In urethane-anaesthetized guinea pigs, intravenous injections of SP derivatives contracted differently bronchial smooth muscle and caused hypotension. The most effective bronchoconstrictor among SP analogs was the gamma-(glutamyl5)Spd derivative of SP (Spd-SP; EC50 = 5.3 nmol/kg), which was more potent than the native peptide (EC50 = 26.5 nmol/kg). In contrast, the gamma-(glutamyl5)Spm derivative of SP (Spm-SP) was found completely unable to cause bronchoconstriction and was significantly less effective than SP in determining hypotension. The contractile effect of Spd-SP and Spm-SP was investigated in vitro on rat isolated colon, a well-characterized preparation rich in NK2 receptors. In addition, Spd-SP was tested on the endothelium-denuded rabbit pulmonary artery (RPA) and the hamster isolated trachea (HT), both tissue preparations containing only a single functional receptor subtype (NK2A and NK2B, respectively). The results obtained showed that Spd-SP recognizes NK2 receptors occurring on rat isolated colon more effectively (EC50 = 11 nM) than the native peptide (EC50 = 45 nM). Conversely, Spm-SP evokes a contractile response less effective than that elicited by SP (EC50 = 312 nM). Furthermore, Spd-SP (0.1-10 microg kg(-1)) produced a concentration-dependent contraction of both HT and RPA, exhibiting a potency respectively 12 and 30 times higher than SP in contracting HT and RPA. Our results indicate that the introduction of a Spd moiety at the level of glutamine-5 of SP gives rise to an analog that possesses a different capability to recognize NK2 receptors than the parent peptide. Moreover, since Spd-SP seems to contract more effectively RPA than HT, we conclude that it preferentially recognizes the NK2A receptor subtype.

Transglutaminase-mediated amine incorporation into substance P protects the peptide against proteolysis in vitro.

The in vitro metabolism of transglutaminase-synthesized substance P analogs has been characterized comparing their stability to that of the parent peptide. The major metabolites have been purified and their structures elucidated by mass spectrometry. Our results demonstrated that gln5 spermidine and spermine analogs of substance P possess an enhanced resistance to the action of proteases. Moreover spermine, a large size hydrophilic compound, specifically prevented the hydrolysis at Phe7-Phe8 bond.

Protective effect of SV-IV on platelet-activating factor-induced hypotension, bronchoconstriction and gastric mucosal injury.

The inhibitory effect of one of the major proteins secreted by the rat seminal vesicles (SV-IV) on platelet-activating factor (PAF)-induced biological activities was investigated in vivo. SV-IV was found to prevent dose dependently both hypotension and acute bronchospasm caused by PAF administration in guinea-pigs. In addition, SV-IV inhibited both PAF- and ethanol-induced gastric mucosal injury in a dose-dependent manner.

[Parasitic metazoans of Stenella coeruleoalba (Cetacea: Delphinidae) stranded along the coast of Latium, 1985-1991]. / Metazoi parassiti di Stenella coeruleoalba (Cetacea: Delphinidae) spiaggiata lungo le coste laziali dal 1985 al 1991.

The striped dolphin represents the most common species of cetacean stranded along the Italian coasts. A parasitological survey on 17 specimens of Stenella coerulecaiba stranded along coasts of Latium from 1985 to 1991, has been carried out. The morphological study enabled the identification of the following parasites. The sites are reported in brackets. DIGENEA: Campula rochebruni (liver), Campula palliata (liver), Pholeter gastrophilus (pyloric stomach). CESTODA: Tetrabothrium forsteri (intestine), Strobilocephalus triangularis (intestine), Monorygma grimaldii, larvae (abdominal cavity, mesentery, testes), Phyliobothrium delphini, larvae (subcutaneous fat). NEMATODA: Skrjabinalius sp. (lungs). COPEPODA: Pennella sp. (skin). ISOPODA: Ceratothoa parallela (mouth, stomach). AMPHIPODA: Syncyamus aequus (blowhole).

[Re-description of Syncyamus aequus, Lincoln and Hurley, 1981 (Amphipoda: Cyamidae), and ectoparasite of Stenella coeruleoalba (Meyen, 1883) and first discovery in Italian waters]. / Ridescrizione di Syncyamus aequus, Lincoln ed Hurley, 1981 (Amphipoda: Cyamidae), ectoparassita di Stenella coeruleoalba (Meyen, 1833) e prima segnalazione per le acque italiane.

Syncyamus aequus Lincoln & Hurley, 1981, an amphipod parasite reported for the first time in South African waters, is re-examined in order to clarify the taxonomic value of some morphological characters not described in detail in the original description and in the following records (Raga, 1988; Sedlak-Weinstein, 1991). Three females (two ovigerous) and six males were collected from two specimens of Stenella coeruleoalba (Meyen, 1833) stranded along the Central Thyrrenian coasts in 1988 and 1993. The amphipods fixed and stored in 70 degrees ethanol were cleared with lactophenol for examination. Drawings were made with the aid of a Leitz microscope drawing attachment. The specimens examined showed the presence of spines located on pereopods I and II, which were lacking in the original description of S. aequus. The authors had the opportunity to compare the specimens (male, female and ovigerous female) with the paratype (PEMK2g) deposited in the Natural History Museum of London. The present re-examination reveals a morphological homogeneity among the specimens collected in Italian waters, the paratype and the individuals recorded by Raga (1988), thus suggesting that they belong to the same species, having a broad geographical distribution. This is the first record of S. aequus in Italian waters.

Populations of acanthocephalus anguillaePomphorhynchus laevis in rivers with different pollution levels

The distribution of two acanthocephalan species (Pomphorhynchus laevisAcanthocephalus anguillae) in the chub (Leuciscus cephalus) was studied in four river reaches characterized by different levels of pollution: the River Ticino near Abbiategrasso (unpolluted), the Naviglio Grande Canal, in Milano (slightly polluted), the River Lambro near Merone village (polluted) and the River Lambro near Monza (severely polluted).Pomphorhynchus laevis was restricted to the unpolluted and the slightly polluted sites, while the intensity of A. anguillae increased proportionally to water pollution. These differences were partially explained by the variation in abundance of their intermediate hosts (Echinogammarus stammeri for P. laevisAsellus aquaticus for A. anguillae). Data on the occurrence of P. laevis and A. anguillae showed a significant negative binomial frequency distribution, suggesting their tendency to be aggregated within the host populations of L. cephalus.

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.

Endoparasites of chub (Leuciscus cephalus) in two rivers of the Abruzzo region of Italy.

The relationship between communities of chub endoparasites (Leuciscus cephalus) fished in the Orta and Pescara Rivers in the Abruzzo region of Italy, and the quality of the water in which they are caught, were studied in surveys designed to evaluate the feed quality of fish in the inland waters of the Abruzzo. Samples were taken monthly from October 2000 to September 2001 in the Orta River (Buscesi District) and the Pescara River (near the Villareia bridge); a total of 86 chub were caught. During periods of low and moderate flow in both rivers, benthonic macroinvertebrates were sampled at the fish sampling sites to classify the water quality using the extended biotic index (EBI) method. The Orta River was moderately polluted and the Pescara River slightly more polluted than the Orta. A parasitological study of the fish was conducted using conventional methods. A morphological study of the parasites led to the identification of seven species of endoparasites. Five of these (Allocreadium isoporum, Caryophyllaeus brachycollis, Caryophyllaeides fennica, Rhabdocona denudata and Pomphorhyncus laevis) were found at both sampling sites, while Acanthocephalus clavula was found only in the Pescara River and Neoechinorhynchus rutili was found only in the Orta River.

Transglutaminase covalently incorporates amines into human immunodeficiency virus envelope glycoprotein gp120 in vitro.

Human immunodeficiency virus envelope glycoprotein gp120, but not its precursor gp160, covalently incorporates both spermidine and glycine ethyl ester in the presence of Ca2+ and transglutaminase purified from guinea pig liver. The examined ability to act as enzyme substrate of various glutamine-containing gp120 fragments, including the principal neutralizing determinant, the CD4 binding domain, and the sequence 254-274, suggested to be involved in post-binding events and in virus entry in the host cell, indicated the glutamine-265 as possible reactive acyl donor site of the protein.

Keratinocyte transglutaminase promoter analysis. Identification of a functional response element.

Keratinocyte transglutaminase catalyzes isopeptide bond formation to yield the highly insoluble cross-linked envelope during terminal differentiation of epidermal cells. Transcriptional response elements were identified in the 5'-flanking DNA of the gene for this enzyme by a combination of transient transfection and electrophoretic mobility shift analyses. Since human keratinocytes transcribed ineffectively transfected transglutaminase flanking DNA, a key feature of these experiments was the use of rat bladder epithelial cells as recipients. Serial deletion experiments identified by transient transfection an important response region containing three putative AP2-like response elements approximately 0.5 kilobases from the transcription initiation site. Oligonucleotides, each containing a single one of the elements, formed specific complexes with keratinocyte nuclear proteins. Two of the response elements were found to be functional by transfection in site-specific deletion experiments. Of these one formed specific DNA-protein complexes with nuclear proteins only from cells exhibiting keratinocyte differentiation. UV cross-linking experiments estimated the protein component of the complex to be approximately 85 kDa. This response element alone increased substantially the transcription of a minimal transglutaminase promoter in transient transfections. Further characterization of the putative transcription factor binding to this response element may provide insight into the regulation of keratinocyte transglutaminase.

Biological activities of a major protein secreted from the rat seminal vesicles after structural modification catalyzed by transglutaminase in vitro.

The immunosuppressive, anti-inflammatory and anti-thrombotic properties of SV-IV, a major protein secreted from the epithelium of rat seminal vesicles, were investigated after transglutaminase-catalyzed covalent incorporation of two molecules of spermidine (Spd) into the protein at the level of Gln-9 and Gln-86. The modified molecular form of the protein (Spd2-SV-IV) showed a more marked inhibitory activity on Con A-induced lymphocyte blastogenesis in comparison with the native protein, whereas no differences in the ability to inhibit the mixed lymphocyte reaction and to decrease the rat epididymal sperm immunogenicity were found between modified and native SV-IV. Spd2-SV-IV was also less effective than native SV-IV to inhibit platelet aggregation induced in vivo by different thrombogenic agents. In contrast, superimposable inhibitory tracings were observed in the in vitro platelet aggregation experiments performed with the two different molecular forms on the protein. Finally, Spd2-SV-IV was shown to retain unchanged the anti-inflammatory activity of native SV-IV.

Human-immunodeficiency-virus transmembrane glycoprotein gp41 is an amino acceptor and donor substrate for transglutaminase in vitro.

Recombinant gp41, the transmembrane glycoprotein of the human-immunodeficiency-virus (HIV) envelope, is an amino acceptor and donor substrate for transglutaminase in vitro. Gln51, Gln52, Gln66 and Lys77 residues were suggested as reactive sites, recognized by the enzyme, for possible cross-linking reactions with gp120, CD4 or other receptor(s) occurring on the surface of HIV-target cells. Soluble CD4, even though unable to function as an amino-acceptor transglutaminase substrate, becomes active in the presence of gp41, negatively influencing the enzyme-catalyzed incorporation of the polyamine spermidine into the transmembrane protein. These results suggest a possible role for transglutaminase in virus entry into host cells, via receptor-mediated endocytosis, and/or in HIV-induced CD4+ T-cell depletion via apoptosis.

Implication of tissue transglutaminase and desmoplakin in cell adhesion mechanism in human epidermis.

The distribution patterns of both tissue and keratinocyte transglutaminases (TGase), as well as that of desmoplakin (DP), have been immunohistochemically investigated in human skin cultured in the absence or presence of cystamine and enalapril, two acantholytic agents. In the control samples, tissue TGase is predominantly expressed in lower layers of the epidermis and is located intercellularly. Conversely, in tissues cultured with cystamine or enalapril, a diffuse cytoplasmatic staining was observed. Similarly, DP, detected on the cell membrane in the control, shifts into the cytosol of the keratinocytes following treatment. The distribution pattern of the keratinocyte enzyme in the acantholytic epidermis was identical to that observed in the normal one. Since cystamine and enalapril are TGase inhibitors and DP was shown to act as a TGase substrate in vitro, we suggest that DP and tissue enzyme may participate in cell adhesion at the intraepidermal level.

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.