Targeted overexpression of Drosophila transglutaminase-B induced characteristic phenotypes in a manner similar to transglutaminase-a.

The Drosophila transglutaminase gene (CG7356) encodes two transglutaminases, dTG-A and dTG-B. To understand the roles of dTG-B during the development of the fly, we examined phenotypes induced through ectopic expression of dTG-B. Overexpression of dTG-B induced rough eye and extra wing crossvein phenotypes. These phenotypes were similar to those observed in the case of targeted overexpression of dTG-A.

Overexpression of transglutaminase in the Drosophila wing imaginal disc induced an extra wing crossvein phenotype.

To determine the roles of Drosophila transglutaminase-A (dTG-A), we examined a phenotype induced through ectopic expression of dTG-A. Overexpression of dTG-A in the wing imaginal disc induced an extra wing crossvein phenotype. This phenotype was suppressed by crossing with epidermal growth factor receptor (Egfr) signaling pathway mutant flies. These results indicate that this phenotype, induced by dTG-A, is related to enhancement of the Egfr signaling pathway.

Over-expression of transglutaminase in the Drosophila eye imaginal disc induces a rough eye phenotype.

Transglutaminases (TGs) catalyze the cross-linking of proteins and are involved in various biological processes in mammals. In invertebrates, except for the involvement in the hemolymph clotting, the functions of TG have not been revealed. Drosophila has a single TG gene (CG7356), from which two kinds of mRNAs (dTG-RA and dTG-RB) are formed. RT-PCR analyses indicated that both dTGs-RA and -RB are synthesized in all the developmental stages tested. To reveal the roles of dTG during the development, we examined a phenotype induced through the ectopic expression of dTG by using a GAL4-UAS targeted expression system. Over-expression of dTG-A in the eye imaginal disc of larva induced a rough eye phenotype in adult compound eyes. Co-expression of P35, an inhibitor of apoptosis, suppressed the rough eye phenotype, suggesting that the rough eye phenotype induced by the over-expression of dTG-A in the eye imaginal disc is due to the occurrence of apoptosis. The rough eye phenotype induced by the over-expression of dTG-A was suppressed by the crossing with mutant fly lines lacking Drosophila JNK gene basket (bsk) or Drosophila JNKK gene hemipterous. FLP-out experiments using an enhancer trap line showed that the over-expression of dTG-A in the eye imaginal disc increased the puckered enhancer activity, a reporter of Bsk activity. These results suggested that the rough eye phenotype induced by the over-expression of dTG-A is related to an enhancement of JNK signaling pathway.

Identification of new amine acceptor protein substrate candidates of transglutaminase in rat liver extract: use of 5-(biotinamido) pentylamine as a probe.

Transglutaminases (TGs) are a family of enzymes that catalyze Ca(2+)-dependent post-translational modification of proteins by introducing protein-protein crosslinks (between specific glutamine and lysine residues), amine incorporation, and site-specific deamidation. In this study, new amine acceptor protein substrates of TG were isolated from rat liver extract and identified using 5-(biotinamido) pentylamine, a biotinylated primary amine substrate, as a probe. TG protein substrate candidates labeled with biotin by endogenous TG activity were isolated and recovered by avidin column chromatography. Proteins with molecular masses of 40, 42, and 45 kDa were the main components of the labeled proteins. Determination of their partial amino acid sequences and immunoblotting analyses were done to identify them. The 45-kDa protein was identical with betaine-homocysteine S-methyltransferase (EC 2.2.2.5), which was identified in our previous study. The 40- and 42-kDa proteins were identified as arginase-I (EC 3.5.3.1) and fructose-1,6-bisphosphatase (EC 3.1.3.11) respectively. TG catalyzed incorporation of 5-(biotinamido) pentylamine into both arginase-I and fructose-1,6-bisphosphatase purified from rat liver was confirmed in vitro. These results suggest that these two enzymes are the new protein substrate candidates of TG and that they can be modified post-translationally by cellular TG.

Comparative biochemical study of N-linked glycans from skin of a squid, Todarodes pacificus.

For comparative biochemical interest, we analyzed the structures of N-glycans in a squid belonging to the Lophotrochozoa, one of the protostome clades. N-Glycans were prepared from squid skin by hydrazinolysis and re-N-acetylation followed by fluorescent tagging with 2-aminopyridine. The labeled N-glycans were purified, and their structures were determined by the two-dimensional HPLC mapping method combined with glycosidase digestions and mass spectrometry. We found that high mannose-type glycans, paucimannose-type glycans and complex-type glycans with a type-1 structure (Galbeta1-3GlcNAc) were dominant in squid skin. The complex-type glycans detected in the squid were similar to those in vertebrates, but have not yet been found in the Ecdysozoa, which is another protostome clade. However, paucimannose-type glycans are commonly found in the Ecdysozoa. Thus, the N-glycan structures of the squid belonging to the Lophotrochozoa have features common to those in vertebrates and the Ecdysozoa including insects and nematodes.

Characterization of wheat germ agglutinin ligand on soluble glycoproteins in Caenorhabditis elegans.

Some mutants of Caenorhabditis elegans show altered patterns of ectopic binding with wheat germ agglutinin (WGA). Some of these mutants also have defects of morphogenesis and movement during development. To clarify the structures of WGA-ligands in C. elegans that may be involved in developmental events, we have analyzed glycan structures capable of binding WGA. We isolated glycoproteins from wild-type C. elegans by WGA-affinity chromatography, and analyzed their glycan structures by a combination of hydrazine degradation and fluorescent labeling. The glycoproteins had oligomannose-type and complex-type N-glycans that included agalacto-biantenna and agalacto-tetraantenna glycans. Although the complex-type glycans carried beta-GlcNAc residues at their non-reducing ends, they did not bind to the WGA-agarose-resin. Thus, it was suggested that these N-glycans were not responsible for WGA-binding of the isolated glycoproteins. Hydrazinolysis of the glycoproteins also released a considerable amount of GalNAc monosaccharide. It was surmised that N-acetylgalactosamine was derived from mucin-type O-glycans with the Tn-antigen structure (GalNAcalpha1-O-Ser/Thr). WGA-blotting assay of neoglycoproteins revealed that a cluster of Tn-antigens was a good ligand for WGA. These results suggested that the WGA-ligand in C. elegans is a cluster of alpha-GalNAc monosaccharides linked to mucin-like glycoprotein(s). The observations reported in this paper emphasize the possible significance of mucin-type O-glycans in the development of a multicellular organism.

Covalent blocking of fibril formation and aggregation of intracellular amyloidgenic proteins by transglutaminase-catalyzed intramolecular cross-linking.

Two different types of physical bonding have been proposed to involve in the formation of neuronal inclusions of patients with neurodegenerative diseases such as Alzheimer's, Parkinson's, and polyglutamine diseases. One is the noncovalent bonding that stabilizes the amyloid-type fibrous aggregates, and the other is the covalent cross-linking catalyzed by tissue transglutaminase. The cross-linking is subdivided into the inter- and intramolecular cross-linking. Little attention has been paid to the pathological roles of the intramolecular cross-linking. To elucidate the possible interplay between the intramolecular cross-linking and the amyloid-type fibril formation, we performed an in vitro aggregation analysis of three intracellular amyloidgenic proteins (a domain of tau protein, alpha-synuclein, and truncated yeast prion Sup35) in the presence of tissue transglutaminase. The analysis was performed in low concentrations of the proteins using techniques including thioflavin T binding and mass spectrometry. The results demonstrated that the amyloid-type fibril formation was strongly inhibited by the transglutaminase-catalyzed intramolecular cross-linking, which blocked both the nucleation and the fiber extension steps of the amyloid formation. Far-UV CD spectroscopy indicated that the cross-linking slightly altered the backbone conformation of the proteins. It is likely that conformational restriction imposed by the intramolecular cross-links has impaired the ordered assembly of the amyloidgenic proteins. Nonamyloid type aggregation was also suppressed by the intramolecular cross-links. On the basis of the results, we proposed that tissue transglutaminase is a modulator for the protein aggregation and can act defensively against the fibril deposition in neurons.

Paradoxical inhibition of protein aggregation and precipitation by transglutaminase-catalyzed intermolecular cross-linking.

Cross-linking of proteins catalyzed by tissue transglutaminase has been suggested to play key roles in a variety of cellular events, including cell apoptosis and human pathogenesis (e.g. polyglutamine and Alzheimer diseases). It has often been suggested that tissue transglutaminase enhances aggregation and precipitation of damaged or pathogenic proteins. To ascertain whether this is accurate, we investigated the effects of tissue transglutaminase-catalyzed modulation on the aggregation of structurally damaged and unfolded proteins. Our results indicated that the aggregation and precipitation of some unfolded proteins were inhibited by transglutaminasecatalyzed reaction, although the effect was strongly dependent upon the target protein species. To elucidate the molecular events underlying the inhibitory effect, extensive analysis was performed with regard to reduced beta-lactoglobulin using a number of techniques, including chromatography and spectroscopy. The results indicated that cross-linking yields high molecular weight soluble polymers but inhibits the growth of insoluble aggregates. The cross-linked beta-lactoglobulin retained stable secondary structures with a hydrophobic core. We concluded that the transglutaminase-catalyzed intermolecular cross-linking did not necessarily enhance protein aggregation but could sometimes have a suppressive effect. The results of the present study suggested that tissue transglutaminase modifies aggregation and deposition of damaged or pathogenic proteins in vivo in a wide variety of manners depending on the target protein species and solution conditions.

In vitro modification of betaine-homocysteine S-methyltransferase by tissue-type transglutaminase.

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. To elucidate the physiological roles of transglutaminase at the molecular level, we need to identify its physiological protein substrates and clarify the relationship between transglutaminase modification of protein substrates and biological responses. Here we examined whether betaine-homocysteine S-methyltransferase (BHMT: EC 2.1.1.5) can be a substrate of tissue-type transglutaminase by in vitro experiments using porcine liver BHMT and guinea pig liver transglutarninase. Guinea pig liver transglutaminase incorporated 5-(biotinamido) pentylamine and [3H] histamine into BHMT in a time-dependent manner. Putrescine and spermidine also seemed to be incorporated into BHMT by transglutaminase. In the absence of the primary amines, BHMT subunits were cross-linked intra- and intermolecularly. BHMT activity was decreased significantly through the cross-linking by transglutaminase. Histamine incorporation slightly reduced the BHMT activity. Peptide fragments of BHMT containing the glutamine residues reactive for transglutaminase reaction were isolated through biotin labelling, proteinase digestion, biotin-avidin a affinity separation, and reverse phase HPLC. The results of amino acid sequence analyses of these peptides and sequence homology alignment with other mammalian liver BHMT subunits showed that these reactive glutamine residues were located in the region near the carboxyl terminal of porcine BHMT subunit. These results suggested that the liver BHMT can be modified by tissue-type transglutaminase and its activity is regulated repressively by the modification, especially by the cross-linking. This regulatory reaction might be involved in the regulation of homocysteine metabolism in the liver.

Identification of an endo-beta-N-acetylglucosaminidase gene in Caenorhabditis elegans and its expression in Escherichia coli.

We report the identification, molecular cloning, and characterization of an endo-beta-N-acetylglucosaminidase from the nematode Caenorhabditis elegans. A search of the C. elegans genome database revealed the existence of a gene exhibiting 34% identity to Mucor hiemalis (a fungus) endo-beta-N-acetylglucosaminidase (Endo-M). Actually, the C. elegans extract contained endo-beta-N-acetylglucosaminidase activity. The putative cDNA for the C. elegans endo-beta-N-acetylglucosaminidase (Endo-CE) was amplified by polymerase chain reaction from the Uni-ZAP XR library, cloned, and sequenced. The recombinant Endo-CE expressed in Escherichia coli exhibited substrate specificity mainly for high-mannose type oligosaccharides. Man(8)GlcNAc(2) was the best substrate for Endo-CE, and Man(3)GlcNAc(2) was also hydrolyzed. Biantennary complex type oligosaccharides were poor substrates, and triantennary complex substrates were not hydrolyzed. Its substrate specificity was similar to those of Endo-M and endo-beta-N-acetylglucosaminidase from hen oviduct. Endo-CE was confirmed to exhibit transglycosylation activity, as seen for some microbial endo-beta-N-acetylglucosaminidases. This is the first report of the molecular cloning of an endo-beta-N-acetylglucosaminidase gene from a multicellular organism, which shows the possibility of using this well-characterized nematode as a model system for elucidating the role of this enzyme.

Inhibition of transglutaminase by synthetic tyrosine melanin.

Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. Previously, we found a high molecular mass transglutaminase-inhibitory substance produced by Streptomyces lavendulae Y-200 that appeared to be a melanin substance. Here, we report that synthetic tyrosine melanin inhibited various types of transglutaminases. Tyrosine melanin inhibited tissue-type transglutaminase in a competitive manner with a glutamine substrate, and also inhibited the cross-linking of casein catalyzed by a tissue-type transglutaminase. The melanized hemolymph of the silkworm and melanin solutions prepared from melanin precursors inhibited tissue-type transglutaminase. These results suggested that the melanin substances generally inhibit transglutaminases.

Identification of mammalian-type transglutaminase in Physarum polycephalum. Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity.

Transglutaminase (TGase) catalyses the post-translational modification of proteins by transamidation of available glutamine residues. While several TGase genes of fish and arthropods have been cloned and appear to have similar structures to those of mammals, no homologous gene has been found in lower eukaryotes. We have cloned the acellular slime mold Physarum polycephalum TGase cDNA using RT-PCR with degenerated primers, based on the partial amino acid sequence of the purified enzyme. The cDNA contained a 2565-bp ORF encoding a 855-residue polypeptide. By Northern blotting, an mRNA of approximately 2600 bases was detected. In comparison with primary sequences of mammalian TGases, surprisingly, significant similarity was observed including catalytic triad residues (Cys, His, Asn) and a GTP-binding region. The alignment of sequences and a phylogenetic tree also demonstrated that the structure of P. polycephalum TGase is similar to that of TGases of vertebrates. Furthermore, we observed that the purified TGase had GTP-hydrolysing activity and that GTP inhibited its transamidating activity, as in the case of mammalian tissue-type TGase (TGase 2).

Method for purification of fluorescence-labeled oligosaccharides by pyridylamination.

We developed a convenient method for purification of PA-oligosaccharides to remove contaminants originating from natural fluorescent materials, and excess reagents as well as by-products of tagging reactions in glycan analysis. The method, using a C18-cartridge, is simple and powerful to remove them. Several examples of experiments that showed the usefulness of this purification method are described in this report.

Structural analysis of N-linked glycans in Caenorhabditis elegans.

Caenorhabditis elegans is an excellent model for morphogenetic research. However, little information is available on the structure of cell-surface glycans in C. elegans, although several lines of evidence have suggested a role for these glycans in cell-cell interactions during development. In this study, we analyzed N-glycan structures. Oligosaccharides liberated by hydrazinolysis from a total membrane fraction were labeled by pyridylamination, and around 90% of the N-glycans were detected as neutral oligosaccharides. The most dominant structure was Man(alpha)1-6(Man(alpha)1-3)Man(beta)1-4GlcNAc(beta)1-4GlcNAc, which is commonly found in insects. Branching structures of major oligomannose-type glycans were the same as those found in mammals. Structures that had a core fucose or non-reducing end N-acetylglucosamine were also identified, but ordinary complex-type glycans with N-acetyllactosamine were not detected as major components.

Co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli.

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of primary amines for the gamma-carboxamide groups of protein bound glutamine residues, and are involved in many biological phenomena. Transglutaminase reactions are also applicable in applied enzymology. Here, we established an expression system of recombinant mammalian tissue-type transglutaminase with high productivity. Overexpression of guinea pig liver transglutaminase in Escherichia coli, using a plasmid pET21-d, mostly resulted in the accumulation of insoluble and inactive enzyme protein. By the expression culture at lower temperatures (25 and 18 degrees C), however, a fraction of the soluble and active enzyme protein slightly increased. Co-overexpression of a molecular chaperone system (DnaK-DnaJ-GrpE) and/or a folding catalyst (trigger factor) improved the solubility of the recombinant enzyme produced in E. coli cells. The specific activity, the affinity to the amine substrate, and the sensitivity to the calcium activation and GTP inhibition of the purified soluble recombinant enzyme were lower than those of the natural liver enzyme. These results indicated that co-overexpression of folding modulators tested improved the solubility of the overproduced recombinant mammalian tissue-type transglutaminase, but the catalytic properties of the soluble recombinant enzyme were not exactly the same as those of the natural enzyme.

A monoclonal antibody to cyclomaltoheptaose (beta-cyclodextrin): Characterization and use for immunoassay of beta-cyclodextrin and its derivatives.

Cyclomaltoheptaose (beta-cyclodextrin, beta-CD) is a promising compound for application in various industrial fields because of its ability to entrap various compounds into its hydrophobic cavity. A monoclonal antibody (A7) to beta-CD was generated by using a conjugate of glucosaminylmaltosyl-beta-CD and bovine serum albumin as an antigen. The A7 monoclonal antibody was IgM/kappa and reacted with beta-CD with high specificity. The epitope recognized by the A7 monoclonal antibody seemed to be located on the secondary hydroxyl groups of the rim side of the beta-CD molecule. The dissociation constant of the complex of beta-CD and the immobilized A7 monoclonal antibody was determined to be 1.2 x 10(-4) M. A competitive ELISA using the A7 monoclonal antibody enabled determination of beta-CD and its derivatives with a detection limit of 0.05 muM. This immunoassay was useful to determine beta-CD in biological fluids such as human plasma and urine after appropriate pretreatment of the samples.