Isolation, sequence analysis, and biological activity of atrolysin E/D, the non-RGD disintegrin domain from Crotalus atrox venom.

Crotalid snake venom metalloproteinases often have associated with them nonproteinase domains that may be processed from the mature proteinases. Nascent atrolysin E, from the western diamondback rattlesnake, Crotalus atrox, has a metalloproteinasedomain and a non-RGD disintegrin domain that is lacking in the mature metalloproteinase. In this studywe report on the isolation, sequence analysis, andbiological activity of the 7.4-kDa atrolysin E disintegrin domain (atrolysin E/D). Atrolysin E/D represents approximately 0.2% of the total protein fromthe crude venom. The protein begins with a glycinyl residue found in the latter part of the spacer region. The sequence of atrolysin E/D is identical to thatof the non-RGD disintegrin domain of atrolysin E. The structure is termed a non-RGD disintegrin sincein lieu of the characteristic RGD sequence, a Met-Val-Asp (MVD) is found instead. Nevertheless, the protein is a potent inhibitor of both collagen- and ADP-stimulated platelet aggregation with IC50 values of 4 and 8 nM, respectively. A cyclized synthetic peptide, Ac-CRVSMVDRNDDTC-NH2, which represents the sequence of the atrolysin E/D non-RGD loop, was demonstrated to be an effective inhibitor of platelet aggregation. Therefore, this region of atrolysin E/D's structure, as in the disintegrins proper, is important for the biological activity of the protein. Thus, like the non-RGD disintegrin barbourin from Sistrurus miliarius barbouri, a RGD sequence in the context of the disintegrin protein backbone is not an absolute requirement for platelet aggregation inhibitory activity. These data underscore the biochemical and functional complexity of crotalid snake venoms due to differential proteolytic processing of the precursor metalloproteinases and exemplify how the processed fragments may contribute to the observed pathological effects of the venom.