Molecular cloning and recombinant expression of a functionally active disintegrinfrom Trimeresurus jerdonii

Sanz, L.¹, Chen, R-Q.², Xiong, Y-L.², Pérez, A¹., Hilario, R.¹, Marcinkiewicz, C.³ & Calvete, J.J.¹

1 Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain; 2 Department of Animal Toxinology, KunmingInstitute of Zoology, The Chinese Academy of Science, Kunming650223, People´sRepublic of China; 3 Temple University College of Science and Technology, Philadelphia, USA

Disintegrins are released in the venoms Crotalidae and Viperidae snakes by proteolytic processing of PII metalloproteinases, and inhibit with high potency and selectivity the interactions of b1 and b3 integrins with their ligands. The integrin inhibitory activity of disintegrins depends on the appropriate pairing of cysteines which determine the conformation of the inhibitory loop. In most single-chain disintegrins the active sequence is RGD. These disintegrins show different binding affinity and selectivity towards integrins which recognise the RGD sequence in their ligands (i.e. a_IIbb3, a_vb3 and a5b1). Dimeric disintegrins exhibit the largest sequence diversity in their integrin binding motifs and include selective and potent antagonist of the integrins a4b1 and a4b7 (MLD motif) and of the integrin a5b1 (MGD, WGD, VGD). On the other hand, obtustatin, purified from the venom of Viperalebetina obtusa, is a potent and selective inhibitor of the a1b1 integrin in vitro and of angiogenesis in vivo. The solution structure and internal motions of obtustatin have been investigated by NMR methods. The active KTS tripeptide of obtustatin is oriented towards a side of its nine residue integrin-binding loop. The C-terminal tail and the integrin-binding loop display a concerted hinge movement articulated at residues located at the base of the loop. Threonine 22, which has been mapped as the most critical residue for blocking the a1b1-integrin collagen IV interaction, is oriented towards the loop center, pointing to its functional importance in maintaining the active conformation of the a1b1 inhibitory loop. To gain further insights into the structure and function of non-RGD short disintegrins, we have cloned the disintegrin jerdostatin from a cDNA library of Trimeresurusjerdonii. Jerdostatin (Q7ZZM2) exhibits 97% amino acid sequence similarity with obtustatin and contains a RTS motif in its integrin-recognition loop. Jerdostatin was cloned as a fusion protein to thioredoxin in a pET32a vector, and was expressed as a soluble product in E. coli BL21. The chimeric protein was purified by Chelating-Sepharose chromatography and its two components were liberated by proteolysis with enteroquinase. Recombinant jerdostatin (cJerd) was purified by FPLC and characterized by N-terminal sequencing and MALDI-TOF mass spectrometry. cJerd contained the expected amino acid sequence and molecular mass (4898 Da), and its 8 cysteine residues were engaged in disulphide bonds. Furthermore, cJerd inhibited the binding of integrin a1b1 to immobilized collagen IV with an IC50 of about 400 nM. Structural (NMR) and functional analyses of native and site-directed mutated cJerd are underway to delineate the contributions of the size, composition, electrostatic potential, and flexibility of the integrin recognition loop and the C-terminal tail to the blocking characteristics toward integrin a1b1.

Correspondence to: libia.sanz@ibv.csic.es or jcalvete@ibv.csic.es