Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease.

Streptomyces mobaraensis secretes a Ca2+-independent transglutaminase (TGase) that is activated by removing an N-terminal peptide from a precursor protein during submerged culture in a complex medium [Pasternack, R., Dorsch, S., Otterbach, J. T., Robenek, I. R., Wolf, S. & Fuchsbauer, H.-L. (1998) Eur. J. Biochem. 257, 570-576]. However, an activating protease could not be identified, probably because of the presence of a 14-kDa protein (P14) belonging to the Streptomyces subtilisin inhibitor family. In contrast, if the microorganism was allowed to grow on a minimal medium, several soluble proteases were extracted, among them the TGase-activating protease (TAMEP). TAMEP was purified by sequential chromatography on DEAE- and Arg-Sepharose and used to determine the cleavage site of TGase. It was clearly shown that the peptide bond between Phe(-4) and Ser(-5) was hydrolyzed, indicating that at least one additional peptidase is necessary to complete TGase processing, even if TAMEP cleavage was sufficient to obtain total activity. Sequence analysis from the N-terminus of TAMEP revealed the close relationship to a zinc endo-protease from S. griseus. The S. griseus protease differs from other members of the M4 protease family, such as thermolysin, in that it may be inhibited by the Streptomyces subtilisin inhibitor. P14 likewise inhibits TAMEP in approximately equimolar concentrations, suggesting its important role in regulating TGase activity.

Enzymatic cross-linking of purple membranes catalyzed by bacterial transglutaminase.

It was found that bacterial transglutaminase (TGase) facilitates selective cross-linking of bacteriorhodopsin (BR) in purple membrane (PM) form under mild conditions. Fluorescent probes were used to detect that the membrane protein BR may act as a glutamine donor as well as a lysine donor for TGase. The binding sites were determined to be Gln-3 as the reactive glutamine, and Lys-129 is the corresponding lysine residue. Upon incubation of PM with TGase, cross-linking of PM patches can be achieved without an additional spacer molecule. To our knowledge, this is the first time that an intermembrane cross-linking of membrane-bound proteins is reported. Furthermore, this finding may provide the ability to achieve covalent linkage of complete purple membrane patches to synthetic polymers.

A sensitive fluorometric assay for tissue transglutaminase.

We have devised a highly sensitive fluorometric well plate assay for tissue transglutaminase that is suitable for multiple kinetic analyses/high-throughput screening of chemical inventories for inhibitors of this enzyme. The procedure measures the rate of fluorescence enhancement (lambda(exc) 260 nm, lambda(em) 538 nm) when 1-N-(carbobenzoxy-l-glutaminylglycyl)-5-N-(5'N'N'-dimethylaminonaphthalenesulfonyl)diamidopentane (glutaminyl substrate) is cross-linked to dansyl cadaverine (amine substrate). The assay procedure can be used to measure the activity of as little as 60 microU of purified guinea pig liver tissue transglutaminase (4.2 ng or 54 fmol of enzyme).

Lysine-rich histone (H1) is a lysyl substrate of tissue transglutaminase: possible involvement of transglutaminase in the formation of nuclear aggregates in (CAG)(n)/Q(n) expansion diseases.

Histone H1, which contains about 27% lysine, is an excellent lysyl donor substrate of Ca(2+)-activated guinea pig liver tissue transglutaminase as judged by rapid fluorescence enhancement in the presence of the glutaminyl-donor substrate 1-N-(carbobenzoxy-L-glutaminylglycyl)-5-N-(5'N'N'-dimethylamino naphth alenesulfonyl) diamidopentane. Sodium dodecyl sulfate gel electrophoresis of a 30-min reaction mixture revealed the presence of fluorescent high-M(r) aggregates, which are also formed when histone H1 is incubated solely with activated tissue transglutaminase. Aggregate formation is even more pronounced when histone H1 is incubated with activated tissue transglutaminase and dimethylcasein (glutaminyl donor only). The findings suggest not only that histone H1 is an especially good lysyl substrate of tissue transglutaminase, but that it is also a glutaminyl substrate. Histone H1 is a good lysyl substrate of transglutaminase purified from Streptoverticillium mobaraense, suggesting that the ability of histone H1 to act as a transglutaminase lysyl substrate is widespread. In agreement with previous studies, it was found that human beta-endorphin is a moderately good substrate of tissue transglutaminase. At least 8 neurodegenerative diseases, including Huntington's disease, are caused by (CAG)(n) expansions in the genome and by an expansion of the corresponding polyglutamine domain within the expressed, mutated protein. Polyglutamine domains are excellent substrates of liver and brain transglutaminases. A hallmark of many of the (CAG)(n)/polyglutamine expansion diseases is the presence of polyglutamine-containing aggregates within the cytosol and nuclei of affected neurons. Transglutaminase activity occurs in both of these compartments in human brain. In future studies, it will be important to determine whether transglutaminases play a role in (1) cross-linking of histone H1 to glutaminyl donors (including polyglutamine domains) in nuclear chromatin, (2) the formation of nuclear aggregates in (CAG)(n)/polyglutamine expansion diseases, (3) DNA laddering and cell death in neurodegenerative diseases and (4) depletion of neuropeptides in vulnerable regions of Huntington's disease brain.

Enzymic preparation of protein G-peroxidase conjugates catalysed by transglutaminase.

Transglutaminases (TGases, EC 2.3.2.13) have proved to be valuable enzymes for site-directed protein coupling via N(epsilon)-(gamma-L-glutamyl)lysine bonds. Their use in conjugate synthesis would overcome many problems caused by chemical reagents. In this approach, we show for the first time that two proteins with different functionalities, namely soybean peroxidase and protein G, can be cross-linked by bacterial TGase with retention of their activities. Soybean peroxidase and protein G were chosen for the enzymic preparation of a bifunctional conjugate among a series of other TGase substrates detected by enzymic incorporation of small fluorescent or biotinylated molecules. The highest yields of conjugate were obtained with a 15-fold excess of peroxidase in phosphate buffer, pH 7.0. Size exclusion chromatography enabled both purification of the conjugates and recovery of the starting materials. Analysis of bifunctionality revealed the coupling of protein G with an average of three peroxidase molecules.

Bacterial pro-transglutaminase from Streptoverticillium mobaraense--purification, characterisation and sequence of the zymogen.

The zymogen of bacterial transglutaminase was found during cultivation of Streptoverticillium mobaraense (DSMZ strain) using rabbit antibodies raised against the active enzyme. Ion-exchange chromatography at pH 5.0 yielded a highly purified pro-enzyme. Structure information was obtained by means of Edman degradation and analysis of PCR amplified nucleotide fragments. The data revealed an excess of negatively charged amino acids in the pro-region resulting in a decreased isoelectric point of the zymogen. Additionally, the new sequence gave rise to some modifications to the previously published hypothetical structure of prepro-transglutaminase derived from genomic DNA [Washizu, K., Ando, K., Koikeda, S., Hirose, S., Matsuura, A., Takagi, H., Motoki, M. & Takeuchi, K. (1994) Biosci. Biotechnol. Biochem. 58, 82-87]. Inactive transglutaminase, which carries an activation peptide of 45 amino acids, has a calculated molecular mass of 42445 Da. Its pro-region provides for both suppression of activity and increased thermostability. Furthermore, it could be shown that the micro-organism produces a protease which cleaves pro-transglutaminase at the C-side of Pro45. Rapid transformation of the mature enzyme also occurs by addition of other proteases. During conversion, 43 and 41 amino acid peptides are released by bovine trypsin and dispase from Bacillus polymyxa, respectively. The detection of endogenous substrates in the murein layer makes discussion of the physiological role of bacterial transglutaminases necessary.

A fluorescent substrate of transglutaminase for detection and characterization of glutamine acceptor compounds.

A fluorescent dipeptide was designed to discover glutamine acceptor proteins of transglutaminase. Starting materials for synthesis were the commercially available compounds carbobenzoxy-L-glutaminylglycine (CBZ-Gln-Gly) and monodansylcadaverine (C-DNS) which were coupled to obtain CBZ-Gln-Gly-C-DNS 1 [1-N-(carbobenzoxy-L-glutaminylglycyl)-5-N- (5'-N', N'-dimethylamino-1'-naphthalenesulfonyl)- diamidopentane]. The glutamine peptide is a substrate of bacterial transglutaminase from Streptoverticillium mobaraense as well as of the guinea pig liver enzyme. This could be shown by incorporating 1 into alpha s1-casein resulting in a significant increase in fluorescence intensity and a concomitant inhibition of cross-linking reaction. Additionally, dipeptide 1 is a useful tool to characterize the specificity of transglutaminase toward small primary amines. We established a sensitive HPLC assay and determined the kinetic parameters of several alkylamines. Hydrolysis of 1 is suppressed in the presence of the nucleophiles as it could be demonstrated with different concentrations of butylamine in semiquantitative studies. Together with labeled primary amines, reagent 1 seems to be a particularly suitable tool for examining acceptor-donor relationships of transglutaminase substrates.

Influence of gelatin matrices cross-linked with transglutaminase on the properties of an enclosed bioactive material using beta-galactosidase as model system.

Transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) from Streptoverticillium mobaraense has been used to stabilize immobilisates produced with beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus oryzae and acid-processed gelatins of different qualities as support. The isopeptide level of N epsilon-(gamma-L-glutamyl)-L-lysine bonds formed by transglutaminase was determined to estimate their influence on the kinetic properties of the enclosed beta-galactosidase. An HPLC procedure using precolumn derivatization of the gelatin hydrolysates with FMOC-chloride was chosen which permits the analysis of cross-linked lysine with satisfactory precision. Depending on the gelatin quality, the degree of cross-links necessary for the transformation of gelatin into an insoluble protein was in the range 0.3-32.3% of the available lysine residues. beta-Galactosidase was entrapped in the gelatin matrices with a yield of 8-46% of the initial activity. Long reaction times for cross-linking were due to low yields rather than to the number of isopeptide bonds. Repeated use of the immobilisates did not lead to an appreciable loss of activity. The Vmax of beta-galactosidase were diminished by immobilization caused by a tighter package of the protein chains rather than by the extent of cross-links, while the obtained Km values of the free enzyme and the immobilisates were quite similar. Also, the pH and temperature of optima of the free enzyme and the gelatin immobilisates differ only slightly. The data suggest that the immobilization procedure only moderately affects the activity of enzymes catalysing the reaction of a small compound if gelatin with high jelly strength is cross-linked in a 10% solution with transglutaminase.

A rapid and simple method for the purification of transglutaminase from Streptoverticillium mobaraense.

Transglutaminase from Streptoverticillium mobaraense was partially purified by ion-exchange chromatography on a weak acid material and hydrophobic chromatography. The separation with a strong acid ion-exchanger produces homogeneous transglutaminase, in a single step and with high yields, directly from the centrifuged and filtered culture fluid of the micro-organism. The procedure reproduced several times could be also carried out on a larger scale with the optimized parameters of the laboratory isolations. The purified enzyme demonstrated good storage stability.