Bdellastasin, a serine protease inhibitor of the antistasin family from the medical leech (Hirudo medicinalis)

Abstract

We have reported earlier the isolation and amino acid composition of bdellin A from medical leech, and characterised it as an inhibitor of trypsin, plasmin and acrosin [Fritz, H., Gebhardt, M., Meister, R. & Fink, E. (1971) in Proceedings of the international research conference on proteinase inhibitors (Fritz, H. & Tschesche, H., eds) pp. 271Ϫ280, Walter de Gruyter, Berlin]. In the present study, one of several chromatographic forms of this inhibitor was isolated from a semi-pure preparation. Elucidation of its amino acid sequence revealed that bdellin A is a member of the antistasin family. Therefore, it was renamed bdellastasin to avoid confusion with bdellin B, which is another trypsin-plasmin inhibitor from the medical leech, but of the Kazal type. Furthermore, a synthetic gene of bdellastasin was constructed, and the protein expressed in Saccharomyces cerevisiae with yields of 29 mg/l. The recombinant bdellastasin was purified by hydrophobic interaction and anion-exchange chromatography. Comparison by mass spectroscopy, far-ultraviolet circular dichroism studies, sequence determination, and inhibition characteristics demonstrated the identity of recombinant and native bdellastasin. The K i values of bdellastasin for inhibition of bovine trypsin and human plasmin are in the nanomolar range; no inhibition was detected for factor Xa, thrombin, tissue kallikrein, plasma kallikrein and chymotrypsin. Circular dichroism analyses indicated that bdellastasin is devoid of secondary-structural elements.Keywords : bdellastasin ; bdellin; gelin; leech; serine protease inhibitor.More than a century ago Haycraft (1884) reported on antico-tors from leeches was described by Seemüller et al. (1977Seemüller et al. ( , 1986. These so-called eglins inhibit strongly neutrophil elasagulant activity in the salivary secretion of the leech Hirudo medicinalis. Markwardt (1955) ascribed this activity to a protein, tase, cathepsin G, chymotrypsin and subtilisin. Shortly afterwards, antistasin, the first member of another group of leech which he called hirudin, and which turned out to be a specific and extremely potent inhibitor of thrombin. Later it was found protease inhibitors, was isolated from the Mexican leech Haementeria officinalis (Tuszynski et al., 1989). that leeches contain a variety of protease inhibitors. Fritz et al. (1969bFritz et al. ( , 1971 discovered that extracts of H. medicinalis contain Antistasin is a cysteine-rich 17-kDa protein with anticoagulant and antimetastatic properties. It exerts its anticoagulant eftwo groups of inhibitors directed against plasmin, trypsin and sperm acrosin. These two groups, named bdellins A and B, were fect by inhibiting highly selectively coagulation factor Xa in a competitive tight-binding manner with an inhibition constant distinguished by their elution behaviour on anion-exchange (K i ) of 0.5 nM (Dunwiddie et al., 1989). The molecule consists chromatography. Bdellins A were not characterised fully at that of 119 amino acid residues. Its primary structure features a twotime. Ami...

Production of Full-Length Human Pre-elafin, an Elastase Specific Inhibitor, from Yeast Requires the Absence of a Functional Yapsin 1 (Yps1p) Endoprotease

“…Using the culture conditions described by Moser et al (19), ϳ10 mg/L of recombinant elafin peptides were consistently recovered in the yeast culture media. This is lower than the 29 mg/L of bdellastasin, a 6-kDa serine protease inhibitor, obtained by Moser et al from an analogous fusion protein also cloned in a pVT plasmid (19).…”

“…Pilot experiments for the detection of recombinant elafin molecules were performed with small volumes of yeast culture grown for 3 days in selective medium. For the purification of large amounts of recombinant elafin peptides, we adopted the growth conditions developed by Moser et al for the production of bdellastasin (19). Briefly, a 3-day preculture in selective medium (100 mL) is diluted to 1 L with a culture medium composed of 0.67% Bacto yeast extract, 0.67% NaH 2 PO 4 , and 2% D-glucose.…”

Eisenstasin, new antistasin family inhibitor from the earthworm

“…Similarly to the other antistasin family inhibitors, eisenstasin I and II feature 3 and 4 internal repeats, respectively, of a 24-29 amino acid sequence. The internal repeat, exhibiting a highly conserved pattern of either the 5-cysteine/2-glycine as in most leech antistasins (Moser et al 1998) or the 6-cysteine/2-glycine as in hydra antistasin (Holstein et al 1992), is a striking feature of the antistasin family. Although the repeats are not identical with regard to length or number, the internal repeats in eisenstasin I and II show a conserved pattern of 6-cysteine/2-glycine, at identical positions between the third and fourth cysteine residues (Fig.…”

“…Amino acid sequence analysis revealed that antistasin is a cysteinerich peptide, featuring internal repeats which contain the conserved 6 cysteine and 2 glycine residues. This suggests that the antistasin gene has evolved via gene duplication (Moser et al 1998). The number of internal repeats depends, however, on the species.…”

Synthetic and Natural Peptides as Antithrombotic Agents—A View on the Current Development

“…therefore, it was renamed bdellastasin to avoid confusion with Bdellin B, which is another trypsin-plasmin inhibitor from the medical leech, but of the Kazal type. Bdelastasin does not manifest inhibitor activity towards Factor Ха, chimotripsin, thrombin, tissue or plasma kalikrein, but its Ki values for inhibition of bovine trypsin are of nanomolar range (64). Steinmetzer et al (1999) reported on the highly potent and specific synthetic thrombin inhibitors derived from the natural polypeptide hirudin which they named hirulogs.…”