Function of Disintegrin-like/Cysteine-rich Domains of Atrolysin A
INHIBITION OF PLATELET AGGREGATION BY RECOMBINANT PROTEIN AND PEPTIDE ANTAGONISTS*
- From the Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 and‡Science Institute, University of Iceland, Reykjavik, Iceland
Abstract
Snake venom hemorrhagic metalloproteinase toxins that have metalloproteinase, disintegrin-like and cysteine-rich domains are significantly more potent than toxins with only a metalloproteinase domain. The disintegrin-like domains of these toxins differ from the disintegrin peptides found in crotalid and viperid venoms by the nature of their different disulfide bond structure and, in lieu of the disintegrins’ signature Arg-Gly-Asp (RGD) integrin binding sequence, there is an XXCD disulfide-bonded cysteinyl sequence in that region. Due to these apparent differences, the contribution to the overall function of the hemorrhagic metalloproteinases by the disintegrin-like domain has been unknown. In this investigation we have expressed in insect cells the disintegrin-like/cysteine-rich (DC) domains of the Crotalus atrox hemorrhagic metalloproteinase atrolysin A and demonstrated that the recombinant protein (A/DC) can inhibit collagen- and ADP-stimulated platelet aggregation. Using synthetic peptides, we have evidence that the region of the disintegrin-like domain that is positionally analogous to the RGD loop of the disintegrins is the site responsible for inhibition of platelet aggregation. For these synthetic peptides to have significant inhibitory activity, the -RSECD- cysteinyl residue must be constrained by participation in a disulfide bond with another cysteinyl residue. The two acidic amino acids adjacent to the middle cysteinyl residue in these peptides are also important for biological activity. These studies emphasize a functional role for the disintegrin-like domain in toxins and suggest structural possibilities for the design of antagonists of platelet aggregation.
Footnotes
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↵* This work was supported by National Institutes of Health Grant GM49042 (to J. W. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: Dept. of Microbiology, Box 441 Jordan Hall, UVA Health Sciences Center, Charlottesville, VA 22908. Tel.: 804-924-0050; Fax: 804-982-2514.
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↵1 The abbreviations used are: SVMPs, snake venom metalloproteinases; Acm, acetoamidomethyl; AcNMPV: A. californica nuclear polyhedrosis virus; ADAMs, a disintegrin-like andmetalloproteinase proteins; A/DC, recombinant disintegrin-like/cysteine-rich protein from atrolysin A; MALD-TOF, matrix assisted laser desorption-time of flight; MDCs,metalloproteinase disintegrin-likecysteine-rich proteins; PAGE, polyacrylamide gel electrophoresis.
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- Received August 19, 1996.
- Revision received March 11, 1997.
