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Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech , Hirudo medicinalis ( thrombosis / antithrombin / recombinant DNA / coagulation )
| Content Provider | Semantic Scholar |
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| Author | Harvey, Richard P. Stefani, Lorraine Schamber, F. Courtney, Michael Tolstoshev, Paul Lecocq, J.-P. |
| Abstract | Cloned cDNAs have been isolated that encode a variant of hirudin, a potent thrombin inhibitor that is secreted by the salivary glands of the medicinal leech, Hirudo medicinals. This variant probably corresponds to a form that has been purified from leech heads but differs in amino acid sequence from the hirudin purified from whole leeches. There are at least three hirudin transcripts detectable in leech RNAs that are different in size, site of synthesis, inducibility by starvation, and relationship to hirudin activity. The new hirudin variant predicted by the cDNA and the heterodisperse transcription products suggest a hirudin protein family. The hirudin cDNA was expressed in Esckerichia coli under the control of the bacteriophage X PL promoter. The recombinant product is biologically active, inhibiting the cleavage by thrombin of fibrinogen and a synthetic tripeptide substrate. Leech hirudin is the most potent natural inhibitor of coagulation known (1-4). A very stable noncovalent 1:1 complex is rapidly and specifically formed with a-thrombin, thereby abolishing its ability to cleave fibrinogen (4). To date there is no evidence that it can interact with other components of the human coagulation cascade (5, 6). Hirudin is a polypeptide of 65 amino acids that is stable to extremes ofpH and heat (4). It contains six cysteine residues grouped in the NH2-terminal half of the protein, an acidic COOH-terminal half, and one sulfated tyrosine (7). A hirudin form with isoleucine at the NH2 terminus was first purified from leech heads (H. medicinalis) (4, 8) in which activity was found to be concentrated in the salivary glands. Subsequently, Bagdy et al. (9) adopted new purification schemes using whole leeches instead of heads, yielding a form with Val-Val as the first two NH2-terminal amino acids. The amino acid sequence of the "whole body form" has been determined by independent groups (8, 10), and valine residues at positions 1 and 2 have been confirmed. Both forms had a specific activity of around 8000-10,000 antithrombin units/mg. However, more recently, Baskova et al. (11) described two distinct hirudins: a highly active form in heads with an Ile-1 NH2 terminus and an inactive form in bodies (pseudohirudin) with a Val-Val NH2 terminus. The potency and specificity of hirudin have generated interest in its possible use as a clinical reagent in treatment of thrombotic diseases, but a detailed pharmacological assessment is prevented by the cost and supply of purified material. In animal studies (12-14), hirudin was shown to be pharmacodynamically inert apart from its anticoagulant activity. It has extremely low toxicity (LD50 > 500,000 antithrombin units/kg in rats; ref. 12), appears to be nonantigenic, and is eliminated almost completely via the kidneys in a biologically active form (12). It is effective in preventing venous thrombosis, vascular shunt occlusion, and thrombin-induced disseminated intravascular coagulation in rats (12). Endotoxin-induced disseminated intravascular coagulation is prevented in newly weaned pigs (15). Purification of large quantities of hirudin from leeches for further clinical testing or eventual clinical use is highly impractical, but this problem can potentially be solved by recombinant DNA technology. In addition, cloning of the gene(s) for hirudin should help to resolve questions about different hirudin forms and possible precursor proteins. Here we report the cloning of a cDNA encoding one variant of H. medicinalis hirudin and its expression in Escherichia coli to yield a biologically active product. MATERIALS AND METHODS Leeches. Live Hirudo medicinalis were purchased from Ricarimpex (Audenge, France) and kept in aerated water containing 0.63 mM NaCl, 0.07 mM CaCl2, 0.05 mM MgSO4, and 0.05 mM KCl at ambient temperature. They were fed on citrate-treated rabbit blood from an inflated porcine bladder. Fed leeches were kept in a separate aquarium. Protein Extracts. Frozen leech segments were homogenized in phosphate-buffered saline (0.1 M Na3PO4, pH 7.0/0.15 M NaCl) with a Polytron homogenizer (Brinkmann), and particulate material was sedimented. For bacterial extracts, cells were disrupted by sonication in TGE buffer (25 mM Tris-HCl, pH 8/50 mM glucose/10 mM EDTA) and cleared by centrifugation. RNA Extraction. Powdered, frozen leech sections were added to lx NETS buffer (0.1 M NaCl/1 mM EDTA/10 mM Tris HCl, pH 7.5/0.5% sodium dodecyl sulfate) containing 50% phenol at 95°C and were homogenized immediately. After a cooling period, phases were separated by centrifugation. The phenol phase was reextracted with 2x NETS, and the aqueous phase was reextracted with phenol. Nucleic acid was precipitated from the pooled aqueous phases with 2 vol of ethanol. After centrifugation, the pellet was dissolved in H20, and the solution was adjusted to 2.5 M LiCl and kept at 4°C overnight. To pellet precipitated RNA, the solution was underlaid with 0.25 vol of 3 M LiCl and centrifuged at 15,000 x g for 10 min at 4°C. The pellet was dissolved in H20 and reprecipitated with ethanol. Hirudin Activity. Antithrombin activity in leech or bacterial extracts was measured in a clotting assay (4) using citrated human platelet-poor plasma as a fibrinogen source or in a colorimetric assay using the thrombin chromogenic substrate Tos-Gly-Pro-Arg-p-nitroanilide (Chromozym TH, Boehringer Mannheim; Tos = tosyl) (7). Standard curves Abbreviation: kb, kilobases. tPresent address: Department of Biochemistry and Molecular Biology, Harvard University, 7 Divinity Avenue, Cambridge MA 02138. §To whom all reprint requests should be addressed. Present address: Biotechnology Australia, Pty Ltd., 28 Barcoo Street, East Roseville, NSW 2069, Australia. 1084 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 83 (1986) 1085 were established with standardized bovine thrombin (Roche, Neuilly-sur-Seine, France) in the case of the clotting assay and with standardized hirudin (a gift from F. Markwardt) in the case of the chromogenic assay. cDNA Cloning. cDNA banks were constructed in pBR322 by standard procedures (16). Screening of banks with oligonucleotides was as described (16), with the stringency of washes for the 48-mer probe being 0.3 M NaCl/0.03 M sodium citrate/0.1% NaDodSO4 at 500C. Oligonucleotides were synthesized by the phosphotriester method on an inorganic support (17). DNA sequence analysis of clones was performed by the dideoxy chain-termination method (18) after subcloning into an M13 vector. Thrombin-Sepharose. Thrombin (61 National Institutes of Health units/mg) was bound to CNBr-activated Sepharose beads (Pharmacia) by using the manufacturer's recommended protocol. For affinity selection ofbacterial hirudin, thrombin-Sepharose was added, in batch, to bacterial extracts so that all activity was bound. The Sepharose was sedimented by gravity, washed twice with excess 0.5 M NaCl, and then eluted with 4 vol of 0.1 M 4-aminobenzamidine/25 mM HCl. All incubations and elutions were at ambient temperature for 10 min. Carrier bovine serum albumin (30 pg/ml) was added to the eluted proteins, and 4-aminobenzamidine was removed by dialysis against 25 mM HCl and then H20. [35S]Cysteinelabeled proteins were analyzed on 15% NaDodSO4/polyacrylamide gels (19) after reduction/denaturation in 2.3% NaDodSO4/6.25% 2-mercaptoethanol at 100°C. |
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| Language | English |
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| Content Type | Text |
| Resource Type | Article |
