Identification of promoter region of guinea pig liver transglutaminase gene.

A 5' flanking region of the guinea pig liver transglutaminase gene was cloned and sequenced. The sequences for TATA box and potential binding sites of some regulatory factors were found in this region. The promoter activity of this region was shown by transfecting its fusion-construct with the chloramphenicol acetyltransferase gene into human hepatoblastoma HepG2 cells.

Nucleotide sequence of rat erythropoietin.

The cDNA for the rat erythropoietin (EPO) has been cloned and sequenced. The deduced amino acid sequence consists of 166 amino acid residues, which has a 79% and 95% homology with human and mouse EPOs, respectively. Many short stretches, highly conserved in primate and rodent EPOs, are found in the 3'-noncoding region when insertions and deletions are taken into consideration.

Analysis of epidermal-type transglutaminase (TGase 3) expression in mouse tissues and cell lines.

In the formation of the cornified cell envelope in the epidermis, epidermal-type transglutaminase (TGase 3) cross-links a variety of structural proteins. However, its expression in other tissue has not been investigated. Furthermore, no cell line expressing TGase 3 has been found. The tissue distribution of TGase 3 in mice was investigated using reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting analyses. TGase 3 mRNA was expressed in the brain, stomach, spleen, small intestine, testis, skeletal muscle and skin. The stomach and testis expressed TGase 3 protein in size similar to that observed in the epidermis. Screening various cell lines, a gastric human cancer cell line, MKN-1 and mouse neuroblast cell line, neuro2a, were found to express TGase 3.

Activity and gene expression of transglutaminase in guinea pig liver during the postnatal growing phase.

During the postnatal growing phase from birth to 7 weeks old, the cytosolic transglutaminase activity of guinea pig liver increased 3.8-fold. The enzyme activity in the particulate fraction increased slightly. Immunoblot analyses showed that the postnatal increase in the activity was correlated with in increase in the enzyme protein. The quantity of mRNA of the liver transglutaminase did not change significantly during the postnatal growing phase examined. These results indicated that transglutaminase may be involved in the postnatal development of guinea pig liver and that the amount of transglutaminase in the postnatal liver may be controlled post-transcriptionally.

Cross-linking of a synthetic partial-length (1-28) peptide of the Alzheimer beta/A4 amyloid protein by transglutaminase.

Cerebral deposits of beta/A4 amyloid protein is a pathologic sign of Alzheimer's disease. A synthetic partial-length (1-28) peptide of this protein contains one glutamine and two lysine residues. Here we show that this peptide can be a substrate of transglutaminase, which catalyzes cross-linking between glutamine and lysine residues in peptides, by demonstrating the formation of multimeric peptides due to the action of this enzyme. A modified (Lys28 to L-norleucine) version of the synthetic peptide was also cross-linked, but another modified version (Lys16 to L-norleucine) was very poorly cross-linked, indicating that Lys16 is involved exclusively in the cross-linking of the partial-length peptide catalyzed by transglutaminase.

Characterization of recombinant mouse epidermal-type transglutaminase (TGase 3): regulation of its activity by proteolysis and guanine nucleotides.

Epidermal-type TGase (TGase 3) is involved in the formation of the cornified cell envelope by cross-linking a variety of structural proteins in the epidermis. Unknown proteases activate this enzyme from the zymogen form by limited proteolysis during epidermal differentiation. It has been difficult to isolate sufficient quantities of native enzymes from tissues for biochemical studies of the properties of TGase 3. In this paper, we circumvented these problems by expressing recombinant full-length mouse TGase 3 in a baculovirus system, and purifying it to homogeneity by successive chromatography and HPLC. Treatment of the purified recombinant protein with dispase, a bacterial protease known to activate zymogens, produced activated TGase 3. The migration of TGase 3 zymogen in SDS-polyacrylamide gel electrophoresis was anomalous when the proTGase 3 was pre-incubated with calcium ion. GTP inhibited the enzymatic activity of recombinant TGase 3. Calpain, a calcium-dependent neutral protease, was a candidate protease, but had no effect on the activation of TGase 3 zymogen.

Molecular cloning and expression of Caenorhabditis elegans ERp57-homologue with transglutaminase activity.

Formation of cross-linking between proteins via a gamma-glutamyl-epsilon-lysine residue is an important process in many biological phenomena including apoptosis. Formation of this linkage is catalyzed by the enzyme transglutaminase, which is widely distributed from bacteria to the animal kingdom. The simple multi-cellular organism Caenorhabditis elegans also possesses transglutaminase activity associated with apoptosis [Madi, A. et al. (1998) Eur. J. Biochem. 253, 583-590], but no gene with significant homology to vertebrate or bacterial transglutaminases has been found in the C. elegans genome sequence database. On the other hand, protein disulfide isomerases were recently recognized as a new family of transglutaminases [Chandrashekar, R. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 531-536]. To identify the molecule with transglutaminase activity in C. elegans, we isolated from C. elegans a gene homologous to ERp57, which encodes a protein disulfide isomerase, expressed it in recombinant form, and characterized the transglutaminase and protein disulfide isomerase activities of the resultant protein. The C. elegans ERp57 protein had both enzyme activities, and the transglutaminase activity had similar characteristics to the activity in lysate of the whole worm. These results suggested that the ERp57 homologue was one of the substances with transglutaminase activity in C. elegans.

Quality deterioration of winged bean during storage.

Seeds of winged bean (Psophocarpus tetragonolobus) were stored under four different sets of conditions; 65% relative humidity (RH) at 5 degrees C, 35% RH at 37 degrees C, 65% RH at 37 degrees C, and 85% RH at 37 degrees C. After storage for three and five months, samples were taken out to explore quality changes during storage. The germination capacity decreased during storage at high temperatures. Although little change was found in content in extractable oil, a decrease in the iodine value and an increase in the acid value and in the TBA value were significant during storage at high temperature and high moisture, indicating deterioration of oil. Aldehyde in defatted bean extract increased under all conditions for storage. Protein extractability was greatly reduced at 36 degrees C and 85% RH, while no reduction was observed at other conditions.

Selective growth of thermostable direct hemolysin-producing Vibrio parahaemolyticus in the alimentary tract of a gastropod, Clithon retropictus, at a selected estuary in Japan.

Thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus increased in cell number in the alimentary tract of a gastropod. Clithon retropictus, from an undetectable level in July to 2.0 x 10(3)/g in September 1991 at the Sada estuary in the southern part of Japan. It was not detected in microalgae, foods of the gastropod, at the estuary or muddy sediments at a neighboring fishing port. The strain was not detected at two estuaries and two fishing ports ecologically separated from the Sada estuary. Serotypes of the isolated strains were different from those registered as clinical isolates. Our results confirm that TDH-producing V. parahaemolyticus grows selectively in the gastropod at some estuaries in Japan.

Ecological cycle of thermostable direct hemolysin-producing strains of Vibrio parahaemolyticus in a brackish-water area with special reference to molluscs and attached microalgae.

Prevalences of thermostable direct hemolysin (TDH)-producing strains in communities of a gastropod mollusc. Clithon retropictus, and a bivalve mollusc, Corbicula japonica, and levels of the strains in attached microalgae and muddy sediments were investigated at a brackish-water area along Hashizu Creek and Togo Pond in Japan, V. parahaemolyticus was detected from attached microalgae at Hashizu Creek in summer months with the highest level of 1.4 x 10(5) cfu/g. Levels of the organism among 20 animals of C. retropictus and C. japonica at the area varied betwen non-detectable level and 10(3) per mollusc in summer months. TDH was detected from culture supernatants of 11-16% of strains isolated from the algae, sediments and C. japonica and 28% of those isolated from C. retropictus at Hashizu Creek. These evidences suggest that C. retropictus would get TDH-positive strains from the algae.

GTP, an inhibitor of transglutaminases, is hydrolyzed by tissue-type transglutaminase (TGase 2) but not by epidermal-type transglutaminase (TGase 3).

Epidermal-type transglutaminase (TGase 3) is devoid of GTPase activity, but its TGase activity is inhibited by GTP as in the case of tissue-type TGase (TGase 2). In addition, the inhibition was not affected by the presence of higher concentrations of Ca ion. These results indicate that GTP interacts with TGase 3 in a manner different from its action on TGase 2.

Expression induced by interleukin-6 of tissue-type transglutaminase in human hepatoblastoma HepG2 cells.

We examined the effect of interleukin-6 (IL-6) on the expression of transglutaminase in human hepatoblastoma HepG2 cells. Treatment of cells with IL-6 increased their transglutaminase activity in a time- and dose-dependent way. Dexamethasone strengthened the stimulation by IL-6. Half-maximum stimulation of transglutaminase activity in the cells occurred at a dose of 40 pM IL-6 regardless of the presence of dexamethasone. Based on its immunoreactivity, the transglutaminase induced was identified as tissue-type transglutaminase. Immunoblot analysis showed that the increase in transglutaminase activity was related to an increase in the amount of transglutaminase protein. Northern blot analysis with a cDNA probe specific for human tissue-type transglutaminase showed that exposure of HepG2 cells to IL-6 increased the mRNA level of the enzyme, and the increase was detectable 3 h after IL-6 was added. Induction of the mRNA was maximum between 10 and 14 h. The increase in the mRNA level was not blocked by the presence of cycloheximide, suggesting that the increase was independent of protein synthesis. Injections into mice of substances that induce inflammation such as turpentine and lipopolysaccharides increased the liver transglutaminase activity. These results indicated that transglutaminase may be involved in some biological processes in hepatocytes regulated by IL-6 signaling.

Determination of amino- and carboxyl-terminal sequences of guinea pig liver transglutaminase: evidence for amino-terminal processing.

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of a variety of primary amines for the gamma-carboxamide groups of protein-bound glutaminyl residues. These enzymes are involved in many biological phenomena. In this study, the amino- and carboxyl-terminal sequences of guinea pig liver transglutaminase were identified by sequence analysis to determine whether this enzyme is processed posttranslationally at its terminal regions. Two peptides, believed to contain the amino-terminal sequences of transglutaminase, were isolated from the Pronase digest of the enzyme protein with SP-Sephadex C-25 column chromatography and reverse-phase HPLC. Analyses (amino acid analysis, sequencing after the treatment with an acylamino-acid-releasing enzyme, and fast atom bombardment mass spectrometry) of these peptides indicated that the amino-terminal structure of this enzyme is acetylAla-Glu-Asp-Leu-Ile-Leu-Glu. The candidate for the carboxyl-terminal peptide in the trypsin digest of enzyme was isolated from the unadsorbed fraction of affinity chromatography with anhydrotrypsin agarose gel. The peptide was found to be Asn-Val-Ile-Ile-Gly-Pro-Ala. Both the terminal sequences were completely consistent with those predicted from the cDNA sequence [Ikura, K., Nasu, T., Yokota, H., Tsuchiya, Y., Sasaki, R., & Chiba, H. (1988) Biochemistry 27, 2898-2905]. These results indicated that the amino-terminal processing occurred after or in the course of translation of this enzyme, namely, removal of the initiator methionine and a subsequent acetylation of the alanine residue adjacent to the methionine. Our results did not indicate carboxyl-terminal processing of guinea pig liver transglutaminase.

Expression of guinea-pig liver transglutaminase cDNA in Escherichia coli. Amino-terminal N alpha-acetyl group is not essential for catalytic function of transglutaminase.

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of a variety of primary amines for the gamma-carboxamide groups of protein-bound glutamine residues. These enzymes are involved in many biological phenomena. Transglutaminase reactions also have been shown to be suitable for applied enzymology. In this study, as a first step of studies to elucidate the structure/function relationship of transglutaminase, we constructed an expression plasmid, pKTG1, containing a cDNA of guinea-pig liver transglutaminase between the NcoI and PstI sites of an expression vector, pKK233-2, and produced the liver transglutaminase as an unfused protein in Escherichia coli. The purified recombinant enzyme was indistinguishable from natural liver transglutaminase in some structural properties such as molecular mass, amino acid composition, and amino- and carboxyl-terminal sequences. However, the alpha-amino group of the amino-terminal alanine residue of the recombinant transglutaminase was not acetylated as was that of the natural enzyme. Comparison of the recombinant enzyme with the natural one did not indicate significant differences in specific activity and apparent Km values for substrates in the histamine incorporation into acetyl alpha s1-casein. The sensitivity to activation by Ca2+ and the rate of catalyzed protein cross-linking were also similar between recombinant and natural transglutaminases. These results indicated that the N alpha-acetyl group in natural liver transglutaminase has not a particular role in the catalytic function of this enzyme.

Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells.

Erythropoietin (Epo), a glycoprotein that regulates the formation of erythrocytes in mammals, was produced in cultured tobacco BY2 cells (Nicotiana tabacum L. cv. Bright Yellow 2) by introducing human Epo cDNA via Agrobacterium tumefaciens-mediated gene transfer. Epo was correctly processed and subsequently penetrated the plasma membrane of tobacco cells. However, it remained attached to the cell wall and was not released into the culture medium. Although Epo produced by tobacco cells was glycosylated with N-linked oligosaccharides, these carbohydrates were smaller than those of the recombinant Epo produced in mammalian cells. Epo produced in tobacco exhibited in vitro biological activities by inducing the differentiation and proliferation of erythroid cells. However, it had no in vivo biological activities. A lectin-binding assay indicated the lack of sialic acid residues in the N-linked oligosaccharides of Epo, suggesting that Epo was removed from the circulation before it reached erythropoietic tissues.