cDNA cloning and functional expression of jerdostatin, a novel RTS-disintegrin from Trimeresurus jerdonii and a specific antagonist of the alpha1beta1 integrin.
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.
Sanz
Libia
L
Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain.
Chen
Run-Qiang
RQ
Pérez
Alicia
A
Hilario
Rebeca
R
Juárez
Paula
P
Marcinkiewicz
Cezary
C
Monleón
Daniel
D
Celda
Bernardo
B
Xiong
Yu-Liang
YL
Pérez-Payá
Enrique
E
Calvete
Juan J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003435:Crotalid Venoms; D003545:Cysteine / Q000032:analysis; D018076:DNA, Complementary / Q000235:genetics*; D019483:Disintegrins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D004220:Disulfides / Q000032:analysis; D005088:Exocrine Glands / Q000737:chemistry; D015870:Gene Expression; D039222:Integrin alpha1beta1 / Q000037:antagonists & inhibitors*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010449:Peptide Mapping; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins; D017841:Trimeresurus / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D003435:Crotalid Venoms; D018076:DNA, Complementary; D019483:Disintegrins; D004220:Disulfides; D039222:Integrin alpha1beta1; D011994:Recombinant Proteins; D014357:Trypsin; D003545:Cysteine