cDNA Cloning and Functional Expression of Jerdostatin, a Novel RTS-disintegrin from Trimeresurus jerdonii and a Specific Antagonist of the α1β1 Integrin

Abstract

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 (1)H 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 alpha(1)-K562 cells to collagen IV with IC(50) values of 180 and 703 nm, respectively. The IC(50) values of conformers 2 of r-jerdostatin-R21 and r-jerdostatin-K21 were, respectively, 5.95 and 12.5 microm. Neither r-jerdostatin-R21 nor r-jerdostatin-K21 showed inhibitory activity toward other integrins, including alpha(IIb)beta(3), alpha(v)beta(3), alpha(2)beta(1), alpha(5)beta(1), alpha(4)beta(1), alpha(6)beta(1), and alpha(9)beta(1) up to a concentration of 24 mum. Although the RTS motif appears to be more potent than KTS inhibiting the alpha(1)beta(1) 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.

High-level production of a candidacidal peptide lactoferrampin in Escherichia coli by fusion expression

“…Many AMPs have been expressed successfully in E. coli using fusion his-tag thioredoxin (Cao et al, 2005;Lai et al, 2005;Sanz et al, 2005;Xu et al, 2006;Shlyapnikov et al, 2008). Trx, an anionic protein with an isoelectric point of 4.67, can effectively neutralize the high cation charge of antimicrobial peptide to stabilize expression system.…”

“…E. coli thioredoxin (12 kDa), a heat-stable and soluble low molecular weight protein in the prokaryotic cytoplasm, is well established as a highly soluble and stable fusion partner for high expression (LaVallie et al, 1993;Tenno et al, 2004). Using fusion his-tag thioredoxin, many AMPs have been expressed successfully in E. coli (Cao et al, 2005;Lai et al, 2005;Sanz et al, 2005;Xu et al, 2006;Shlyapnikov et al, 2008). Production of a tag-free protein is especially important for the manufacture of therapeutic antimicrobial peptides.…”

KTS and RTS-Disintegrins: Anti-Angiogenic Viper Venom Peptides Specifically Targeting the α1β 1 Integrin

“…3). This disintegrin has only been characterized as a recombinat protein cloned from a venom gland cDNA library of the chinese Jerdon´s pit viper Trimeresurus jerdonii, and expresses an active RTS tripeptide and the C-terminal dipeptide 42 NG 43 which is removed from the venom-secreted KTS-disintegrins [59]. Wild-type (R 21 ) and the R 21 K mutant are selective inhibitors of the binding of the 1 1 integrin to collagen IV (Fig.…”

CHAPTER 5. Reptile Venoms as a Platform for Drug Development

“…[73][74][75][76] Depending on the active tripeptide motif, the various disintegrins can interact with specic subtypes of integrin receptors, thereby inhibiting the corresponding biological activity ( Figure 5.2). Although most disintegrins in snake venoms are monomers, some are found as homo-or heterodimers.…”