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J. Biol. Chem., Vol. 280, Issue 49, 40714-40722, December 9, 2005

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cDNA Cloning and Functional Expression of Jerdostatin, a Novel RTS-disintegrin from Trimeresurus jerdonii and a Specific Antagonist of the ₁₁ Integrin^*

Libia Sanz1, Run-Qiang Chen1, Alicia Pérez, Rebeca Hilario, Paula Juárez, Cezary Marcinkiewicz¶, Daniel Monleón||, Bernardo Celda||, Yu-Liang Xiong, Enrique Pérez-Payá**, and Juan J. Calvete2

From the Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain, the Department of Animal Toxinology, Kumming Institute of Zoology, The Chinese Academy of Sciences, Kumming 650223, Peoples Republic of China, the ^¶College of Science and Technology, Center for Neurovirology and Cancer Biology, Temple University, Philadelphia, Pennsylvania 19122, the ^||Departamento de Química Física, Universitat de València, Dr. Moliner 50, 46100 Valencia, Spain, and the ^**Centro de Investigación "Principe Felipe" and Consejo Superior de Investigaciones Científicas, Avda. del Saler 16, 46013 Valencia, Spain

Jerdostatin represents a novel RTS-containing short disintegrin cloned by reverse transcriptase-PCR from the venom gland mRNA of the Chinese Jerdons pit viper Trimeresurus jerdonii. The jerdostatins precursor cDNA contained a 333-bp open reading frame encoding a signal peptide, a pre-peptide, and a 43-amino acid disintegrin domain, whose amino acid sequence displayed 80% identity with that of the KTS-disintegrins obtustatin and viperistatin. The jerdostatin cDNA structure represents the first complete open reading frame of a short disintegrin and points to the emergence of jerdostatin from a short-coding gene. The different residues between jerdostatin and obtustatin/viperistatin are segregated within the integrin-recognition loop and the C-terminal tail. Native jerdostatin (r-jerdostatin-R21) and a R21K mutant (r-jerdostatin-K21) were produced in Escherichia coli. In each case, two conformers were isolated. One-dimensional ¹H NMR showed that conformers 1 and 2 of r-jerdostatin-R21 represent, respectively, well folded and unfolded proteins. The two conformers of the wild-type and the R21K mutant inhibited the adhesion of ₁-K562 cells to collagen IV with IC₅₀ values of 180 and 703 nM, respectively. The IC₅₀ values of conformers 2 of r-jerdostatin-R21 and r-jerdostatin-K21 were, respectively, 5.95 and 12.5 µM. Neither r-jerdostatin-R21 nor r-jerdostatin-K21 showed inhibitory activity toward other integrins, including _IIb3, _v3, ₂₁, ₅₁, ₄₁, ₆₁, and ₉₁ up to a concentration of 24 µM. Although the RTS motif appears to be more potent than KTS inhibiting the ₁₁ integrin, r-jerdostatin-R21 is less active than the KTS-disintegrins, strongly suggesting that substitutions outside the integrin-binding motif and/or C-terminal proteolytic processing are responsible for the decreased inhibitory activity.

Received for publication, September 6, 2005

^* This work was supported in part by National Institutes of Health Grant RO1 CA100145-01A1 and American Heart Association Grant 0230163N (to C. M.), and Ministerio de Educación y Ciencia, Madrid, Spain, Grant BFU2004-01432/BMC (to J. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 34-96-339-1778; Fax: 34-96-369-0800; E-mail: jcalvete{at}ibv.csic.es.

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