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Jararhagin ECD-Containing Disintegrin Domain: Expression in Escherichia Coli and Inhibition of the Platelet–Collagen Interaction

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Jararhagin ECD-Containing Disintegrin Domain: Expression in Escherichia Coli and Inhibition of the Platelet–Collagen Interaction Archives of Biochemistry and Biophysics Vol. 369, No. 2, September 15, pp. 295–301, 1999 Article ID abbi.1999.1372, available online at http://www.idealibrary.com on Jararhagin ECD-Containing Disintegrin Domain: Expression in Escherichia coli and Inhibition of the Platelet–Collagen Interaction Ana M. Moura-da-Silva,*,†,‡,1 Alex Lı´nica,* Maisa S. Della-Casa,* Aura S. Kamiguti,§ Paulo L. Ho,¶ Julian M. Crampton,† and R. David G. Theakston‡ *Laborato´rio de Imunopatologia and ¶Laborato´rio de Biotecnologia Molecular, Instituto Butantan, Av. Vital Brasil, 1500, 05503-900, Sa˜o Paulo, Brazil; §Department of Haematology, University of Liverpool, Prescot Street, Liverpool, L69 3BX, United Kingdom; and †Molecular Genetics Unit and ‡Alistair Reid Venom Research Unit, Liverpool School of Tropical Medicine, Pembroke Place, L3 5QA, Liverpool, United Kingdom Received May 17, 1999, and in revised form July 6, 1999 demonstrating that these antibodies recognize the Jararhagin, a hemorrhagin from Bothrops jararaca parent jararhagin molecule. Treatment of the fusion venom, is a soluble snake venom component compris- protein with enterokinase, followed by further cap- ing metalloproteinase and disintegrin cysteine-rich ture of the enzyme, resulted in a band of 30 kDa, the domains and, therefore, is structurally closely related expected size for jararhagin-C. Further purification of to the membrane-bound A Disintegrin And Metallo- the cleaved disintegrin using FPLC Mono-Q columns proteinase (ADAMs) protein family. Its hemorrhagic resulted in one fraction capable of efficiently inhibit- activity is associated with the effects of both metallo- ing collagen-induced platelet aggregation in a dose- proteinase and disintegrin domains; the metallopro- dependent manner (IC50 of 8.5 mg/ml). © 1999 Academic Press teinase enzymatically damages the endothelium and Key Words: disintegrin; metalloproteinase; expres- the disintegrin domain inhibits platelet–collagen in- sion in E. coli; venom; platelet. teractions. The expression of whole jararhagin or its disintegrin domain has never been attempted before. The aim of this study was to investigate whether we could express the disintegrin domain of jararhagin The action of snake venom toxins implies binding to and to verify whether this domain displays an inhibi- a variety of critical physiological targets interfering tory effect on the platelet–collagen interaction. There- with important processes such as neuromuscular fore, the cDNA fragment coding for the disintegrin transmission or hemostasis. One particularly impor- plus cysteine-rich domains of jararhagin was cloned tant group of snake venom toxins is the hemorrhagins. into the pET32a vector, used to transform the Esche- These are found mainly in viper venoms and are re- richia coli AD494(DE3)pLysS strain. The thioredoxin– sponsible for the often massive hemorrhage due to the disintegrin fusion protein was recovered from the sol- uble extract of the cells, yielding up to 50 mg/liter disruption of blood vessels as well as for inhibition of culture. The fusion protein was isolated using polyhis- platelet aggregation (1). Hemorrhagins are soluble tidine binding resin which resulted in a main band of zinc-dependent metalloproteinases possessing an addi- 45 kDa recognized by anti-native jararhagin antibod- tional disintegrin-like domain extended by a cysteine- ies. Antibodies raised in rabbits against the fusion rich carboxy terminus, characteristic of the P-III snake protein had high enzyme-linked immunosorbent assay venom metalloproteinases (2). The molecular mecha- titers against native jararhagin and detected a band of nisms related to the activity of the metalloproteinase 52 kDa on Western blots of whole B. jararaca venom domain are well understood and resemble those of the matrixins (3). However, little is known about the action of the disintegrin domain of the metalloproteinases. 1 To whom correspondence should be addressed at Laborato´rio de Imunopatologia, Instituto Butantan, Av. Vital Brasil, 1500-Butan- The most frequently studied disintegrins are the P-II tan, 05503-900, Sao Paulo, SP, Brazil. Fax: 55-11-815.1505. E-mail: snake venom metalloproteinases known as RGD-disin- [email protected]. tegrins (4). These are coded as precursor molecules 0003-9861/99 $30.00 295 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 296 MOURA-DA-SILVA ET AL. with a similar domain orientation to the P-III metallo- merase, and T4 ligase were supplied by Boehringer-Mannheim. proteinases. However, the mature proteins are short pET32 vector, E. coli strains, enterokinase and an enterokinase peptide chains comprising only the disintegrin domain. capture kit, and poly-histidine-binding resin were purchased from a b Novagen (Cambridge, UK). Oligonucleotides were synthesized by They bind to the platelet IIb 3 integrin with nanomo- Genosys (Cambridge, UK). lar affinity, blocking fibrinogen binding and thus inhib- pET32-JD9. The cDNA sequence coding for the disintegrin plus iting platelet aggregation by this route (4). Other func- cysteine-rich domains of jararhagin was amplified by PCR using the tional activities include interference with cell metasta- complete original clone (BJD4) of jararhagin cDNA as template (9; sis in experimental models (5). These activities are EMBL X68251). A SacI site and codons for the amino acid sequence 9 9 directly related to the presence of an RGD motif, which LGTDIISP flanked the amplification product at the 5 end. The 3 end consisted of the anticodons for VATAY amino acids followed by is present on the surface of the molecule in a disulfide a stop codon and a NotI site. These amino acid sequences comprise bond-stabilized loop (6). The P-II and P-III disintegrins respectively the N-terminal residues of the disintegrin domain and share a high amount of sequence similarity. However, the C-terminal residues of the cysteine-rich domain. The PCR prod- the latter possess a carboxy-terminal extension rich in ucts were digested by SacI and NotI and ligated to the same sites of cysteines and they have distinct RSECD motifs, which the linearized pET32a vector. The resulting vector, pET32-JD9, pre- dicts a protein with 109 amino acids of thioredoxin A, a histidine tag, replace the RGD sequence (7). a thrombin site, and an enterokinase site, followed by the disintegrin Venom metalloproteinases and disintegrins share plus cysteine-rich extension of jararhagin. Correct cloning was con- strong sequence similarity to the recently character- firmed by partial sequence of the 59 end of 300 bp, which confirmed ized ADAM2 (A Disintegrin And Metalloproteinase) the complete sequence of the disintegrin domain. family of mammalian proteins (8). The ADAMs have E. coli cultures and protein expression. pET32-JD9 was trans- the same domain organization as venom metallopro- fected into E. coli AD494(DE3)pLysS and cultured at 37°C in Luria– Bertani medium, containing the appropriated antibiotics, to A600 5 teinases added to a transmembrane domain and a po- 0.500. The T7 RNA polymerase under lacUV5 promoter was induced tential signaling cytoplasmic tail. These proteins have with 1 mM isopropyl-thio-b-D-galactopyranoside (IPTG). been ascribed adhesive and antiadhesive properties; Protein isolation. Bacteria from cultures were collected by cen- adhesive properties include sperm–egg fusion as well trifugation, suspended in 1/20 vol of 50 mM Tris/HCl, 500 mM NaCl, as muscle fusion and antiadhesive properties such as 5 mM imidazole, pH 7.9, and sonicated. The soluble intracellular inhibition of neural cell fate at different stages of em- fraction was added to a histidine-binding nickel–Sepharose resin. Elution was performed after extensive washing with 60 mM imid- bryogenesis and axonal extension in Drosophila (8). azole by addition of 1 M imidazole, both in the same Tris buffer. The Therefore, the soluble venom metalloproteinases eluted sample was dialyzed against 20 mM Tris/HCl, 50 mM NaCl, 2 would be expected to possess antiadhesive properties mM CaCl2, pH 7.4, and the recombinant disintegrin released from inhibiting intercellular communication. With native the thioredoxin peptide by digestion with enterokinase (1 U/100 mg jararhagin we have shown that a b integrin is the fusion protein). The cleavage enzyme was then removed using a 2 1 capture kit according to the manufacturer’s instructions. In some selective target of this enzyme (1). However, knowl- experiments, the cleaved fusion protein was further chromato- edge is lacking on the structure of the ECD-containing graphed in FPLC Mono-Q columns (Pharmacia) equilibrated with 20 disintegrin domain, which could explain the specificity mM Tris/HCl, pH 8.0. Retained proteins were eluted with a linear of the integrin binding. It appears therefore that the gradient of 0–1 M NaCl in the same buffer. Protein concentration P-III disintegrins may bind to different types of cell was estimated using Bradford reagent (Amresco, U.S.A.). surface receptors; snake venom peptides could act as Immunochemical analyses. ELISA and Western blotting were carried out using standard protocols (11). Antibodies against the antagonists of the mammalian ADAMs or even ago- native jararhagin (JAR) isolated from B. jararaca venom (9), the nists in integrin-mediated cell activation. recombinant fusion protein (Trx-JD9), and the expression product of Production of recombinant P-III disintegrins is the wild vector (Trx) were raised in rabbits by immunization with therefore crucial for characterizing the molecular 500 mg of each protein in Freund’s complete adjuvant. After 4 weeks, m mechanisms of action of these peptides. In this paper the animals received two boosters of 100 g of samples in saline at 15-day intervals. The antibody titers were assayed by ELISA using we report the successful expression of the disintegrin peroxidase-labeled anti-rabbit IgG conjugates (Sigma). For SDS– domain of jararhagin, a P-III snake venom metallopro- PAGE (12), pellets from 0.5-ml cultures or 10 mg of purified proteins teinase isolated from Bothrops jararaca venom (9) us- (4 mg for pool 6) were diluted in 25 ml of sample buffer (125 mM ing Escherichia coli expression vectors.
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