Dendroaspin: A potent integrin receptor inhibitor from the venoms of Dendroaspis viridis and D. jamesonii

In the last five years the identification of numerous adhesion receptors, and the recognition motifs within their physiological glycoprotein ligands, have elucidated some of the basic principles underlying cell-cell and cell-matrix interactions (1). Most adhesion receptors belong to the integrin supergene family, eg. a11bP3, avP3 (2). A key mechanism common to a number of integrin-ligand interactions involves the recognition of short aspartate-containing sequences or motifs (3), which include the ubiquitous sequence Arg-Gly-Asp (RGD). In this regard, a major breakthrough has been the discovery of a family of small (6-9kDa). RGD-containing polypeptides derived from the venoms of pit vipers, potent inhibitors of integrin-ligand interactions and thus termed disintegrins (4,5,6,7). In the present study we have identified, purified and sequenced a potent, novel disintegrin-like polypeptide from the venom of D.viridis, and an identical polypeptide from D.jamesonii. This polypeptide was originally described as a nontoxic neurotoxin (8) and was identified by us as containing an RGD motif during a search of the Protein Identification Resource (PIR), NBRF, Washington. The RGD motif was found to be held within the 13 amino acid-loop CFTPRCDMPGPYC, which is highly similar to that from the disintegrin, kismn CRIPRGDMPDDRC (9). whereas the remainder of the molecule showed high sequence identiry with the short-chain neurotoxins pable 1.). We have developed a single step reverse phase HPLC purification of dendroaspin (Fig.1). from the whole venoms of Dendroaspis viridis and D. jamesonii kaimosae (Latoxan, France), by chromatography on a Vydac C-18 reverse phase column (6). A single active fraction was identified for each venom (Figl.). Active fractions were identified by testing for the inhibition of ADPinduced fibrinogen-mediated platelet aggregation in platelet rich plasma (PRP) as previously described (6). The amino acid sequence, as published by Joubert et a1 (8), was confirmed using established techniques (10). Table 1. illustrates the degree of homology with the neurotoxin erabutoxin b and the disintegrins, albolabrin and elegantin (6). There is minimal homology with the disintegrins outside the RGD-loop and minimal homology with the neurotoxins within the loop. Isoelectric focusing experiments were performed using PAG (Phmacia) over the ranges pH 5.5-8.5 and pH 3.5-9.5. Running