Christiane Noeske-Jungblut

TRIABIN, A HIGHLY POTENT EXOSITE INHIBITOR OF THROMBIN

Triabin, a new thrombin inhibitor, has been purified from the saliva of Triatoma pallidipennis, a blood-sucking triatomine bug. It forms a noncovalent complex with thrombin at a molar ratio of 1:1, inhibits thrombin-induced platelet aggregation, and prolongs thrombin clotting time and activated partial thromboplastin time. However, it only minimally suppresses the amidolytic activity of thrombin, as measured by a chromogenic peptide substrate assay. It completely blocks trypsin-catalyzed cleavage of thrombin, probably via protection of the anion-binding exosite and inhibits the effect of thrombomodulin on thrombin in a dose-dependent fashion. These results indicate that the inhibitor is directed toward the anion-binding exosite of thrombin. The protein was partially sequenced and the information used to isolate cDNA clones from a T. pallidipennis salivary gland library. Four slightly polymorphic variants coding for mature proteins of 142 amino acids preceded by a putative leader sequence were obtained. The recombinant protein expressed in the periplasmic space of Escherichia coli has a biological activity similar to that of salivary triabin, as tested in a thrombin-induced platelet aggregation assay. In addition, recombinant triabin inhibits thrombin-catalyzed hydrolysis of fibrinogen with a K of about 3 pM.

Rapid effects of EGF on cytoskeletal structures and adhesive properties of highly metastatic rat mammary adenocarcinoma cells

In the highly metastatic rat mammary adenocarcinoma cell clone MTLn3, EGF induced increased adhesion to fibronectin while in the human epidermoid carcinoma cell line A431 EGF induced diminished adhesive properties. Flattening of cells with extensive formation of fllopodia was observed in MTLn3 cells within 5 min of EGF addition, while in A431 cells EGF induced rounding up and only occasional formation of filopodia. Immunofluorescent analysis revealed extension of microtubutes (MT) into the filopodia and Western blot analysis demonstrated an EGF-induced 2- to 3-fold increase in the amount of assembled tubulin in MTLn3 but not in A431 cells. In MTLn3, but only marginally in A431 cells, EGF treatment resulted in phosphorylation of a 280 kD cytoskeleton-associated protein, which was rapid and dose-dependent. These results suggest differential signal transduction pathways of cytoskeleton-associated EGFRs in highly metastatic MTLn3 as compared with A431 cells.

Cloning sequencing and expression of the gene for cytochrome P450meg, the steroid-15?-monooxygenase from Bacillus megaterium ATCC 13368

A 4.3 kb EcoRI fragment carrying the gene for cytochrome P450meg, the steroid-15β-monooxygenase from Bacillus megaterium ATCC 13368, was cloned and completely sequenced. The gene codes for a protein of 410 amino acids and was expressed in Escherichia coli and B. subtilis. Protein extracts from the recombinant E. coli strains were able to hydroxylate corticosteroids in the 15β position when supplemented with an extract from a P450- mutant of B. megaterium ATCC 13368 as a source of megaredoxin and megaredoxin reductase. In contrast, 15β-hydroxylation was obtained in vitro and in vivo without the addition of external electron transfer proteins, when cytochrome P450meg was produced in B. subtilis 168. Protein extracts from nonrecombinant B. subtilis 168 could also support the in vitro hydroxylation by cytochrome P450meg produced in E. coli.

Expression of Triabin, a Novel Highly Potent Inhibitor of Thrombin, in Sf9 and High Five Insect Cells using a Recombinant Baculovirus

Triabin is a novel thrombin inhibitor originally isolated from the saliva of the hematophageous bug Triatoma pallidipennis. The mechanism of thrombin inhibition by triabin is different from other known inhibitors like hirudin or heparin/anti-thrombin III, but the potency of triabin is similar to that of hirudin. Using the cloned cDNA of triabin, a recombinant baculovirus expressing this secreted protein was constructed. However, only low expression rates were obtained in Sf9 insect cells infected with this virus (1–3 mg per 1 culture). Therefore, the expression of triabin in High five insect cells was also investigated and it was found that from these cells 3–8 times higher yields could be obtained. Recombinant triabin purified from the insect culture supernatants had a biological activity similar to that of natural salivary triabin. The availability of an efficient production process and the unique biological properties of triabin suggest that this protein has a potential as a novel therapeutic.