Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Jul 1;357(Pt 1):57–64. doi: 10.1042/0264-6021:3570057

Positional importance of Pro53 adjacent to the Arg49-Gly50-Asp51 sequence of rhodostomin in binding to integrin alphaIIbbeta3.

C P Chang 1, J C Chang 1, H H Chang 1, W J Tsai 1, S J Lo 1
PMCID: PMC1221928  PMID: 11415436

Abstract

Rhodostomin (RHO), a disintegrin isolated from snake venom, has been demonstrated to inhibit platelet aggregation through interaction with integrin alphaIIbbeta3, but there is a lack of direct evidence for RHO-integrin alphaIIbbeta3 binding. In addition, no study on the length of Arg(49)-Gly(50)-Asp(51) (RGD) loop of RHO influencing on its binding to integrin alphaIIbbeta3 has been reported. In the present study we have developed a highly sensitive dot-blot and glutathione S-transferase-RHO pull-down assays; the latter was coupled with a biotin-avidin-horseradish peroxidase enhanced-chemiluminescence detection system. These were able to demonstrate the direct binding of RHO to integrin alphaIIbbeta3. The pull-down assay further showed that four alanine-insertion mutants upstream of the RGD motif and three insertions downstream of the RGD were able to decrease integrin alphaIIbbeta3 binding activity to only a limited extent. By contrast, two insertions immediately next to RGD and one insertion in front of the Cys(57) caused almost complete loss of binding activity to alphaIIbbeta3. The results of the platelet-aggregation-inhibition assay and platelet-adhesion assay for the insertion mutants were consistent with results of the pull-down assay. It is thus concluded that, although an insertion of a single alanine residue in many positions of the RGD loop has only minor effects on RHO binding to integrin alphaIIbbeta3, the specific position of Pro(53) residue adjacent to the RGD sequence is important for RHO binding to platelet integrin alphaIIbbeta3.

Full Text

The Full Text of this article is available as a PDF (322.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adler M., Lazarus R. A., Dennis M. S., Wagner G. Solution structure of kistrin, a potent platelet aggregation inhibitor and GP IIb-IIIa antagonist. Science. 1991 Jul 26;253(5018):445–448. doi: 10.1126/science.1862345. [DOI] [PubMed] [Google Scholar]
  2. Blobel C. P., White J. M. Structure, function and evolutionary relationship of proteins containing a disintegrin domain. Curr Opin Cell Biol. 1992 Oct;4(5):760–765. doi: 10.1016/0955-0674(92)90098-w. [DOI] [PubMed] [Google Scholar]
  3. Calvete J. J. Platelet integrin GPIIb/IIIa: structure-function correlations. An update and lessons from other integrins. Proc Soc Exp Biol Med. 1999 Oct;222(1):29–38. doi: 10.1111/j.1525-1373.1999.09993.x. [DOI] [PubMed] [Google Scholar]
  4. Chang H. H., Hu S. T., Huang T. F., Chen S. H., Lee Y. H., Lo S. J. Rhodostomin, an RGD-containing peptide expressed from a synthetic gene in Escherichia coli, facilitates the attachment of human hepatoma cells. Biochem Biophys Res Commun. 1993 Jan 15;190(1):242–249. doi: 10.1006/bbrc.1993.1037. [DOI] [PubMed] [Google Scholar]
  5. Chang H. H., Lin C. H., Lo S. J. Recombinant rhodostomin substrates induce transformation and active calcium oscillation in human platelets. Exp Cell Res. 1999 Aug 1;250(2):387–400. doi: 10.1006/excr.1999.4547. [DOI] [PubMed] [Google Scholar]
  6. Chang H. H., Tsai W. J., Lo S. J. Glutathione S-transferase-rhodostomin fusion protein inhibits platelet aggregation and induces platelet shape change. Toxicon. 1997 Feb;35(2):195–204. doi: 10.1016/s0041-0101(96)00121-3. [DOI] [PubMed] [Google Scholar]
  7. Chang H.-H., Chang C.-P., Chang J.-C., Dung S.-Z., Lo S.J. Application of Recombinant Rhodostomin in Studying Cell Adhesion. J Biomed Sci. 1997;4(5):235–243. doi: 10.1007/BF02253423. [DOI] [PubMed] [Google Scholar]
  8. Chang T. C., Kou S. H., Chen F. W., Chang C. C., Liaw K. Y., Chang-Chien Y., Huang T. S., Lin C. C., How S. W. Cytopathology of thyroid cancer by fine needle aspiration with Liu's stain. Taiwan Yi Xue Hui Za Zhi. 1982 Nov;81(11):1404–1412. [PubMed] [Google Scholar]
  9. Dennis M. S., Carter P., Lazarus R. A. Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: analysis by site-directed mutagenesis. Proteins. 1993 Mar;15(3):312–321. doi: 10.1002/prot.340150308. [DOI] [PubMed] [Google Scholar]
  10. Dennis M. S., Henzel W. J., Pitti R. M., Lipari M. T., Napier M. A., Deisher T. A., Bunting S., Lazarus R. A. Platelet glycoprotein IIb-IIIa protein antagonists from snake venoms: evidence for a family of platelet-aggregation inhibitors. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2471–2475. doi: 10.1073/pnas.87.7.2471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Du X. P., Plow E. F., Frelinger A. L., 3rd, O'Toole T. E., Loftus J. C., Ginsberg M. H. Ligands "activate" integrin alpha IIb beta 3 (platelet GPIIb-IIIa). Cell. 1991 May 3;65(3):409–416. doi: 10.1016/0092-8674(91)90458-b. [DOI] [PubMed] [Google Scholar]
  12. Fässler R., Georges-Labouesse E., Hirsch E. Genetic analyses of integrin function in mice. Curr Opin Cell Biol. 1996 Oct;8(5):641–646. doi: 10.1016/s0955-0674(96)80105-0. [DOI] [PubMed] [Google Scholar]
  13. Gould R. J., Polokoff M. A., Friedman P. A., Huang T. F., Holt J. C., Cook J. J., Niewiarowski S. Disintegrins: a family of integrin inhibitory proteins from viper venoms. Proc Soc Exp Biol Med. 1990 Nov;195(2):168–171. doi: 10.3181/00379727-195-43129b. [DOI] [PubMed] [Google Scholar]
  14. Gumbiner B. M. Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell. 1996 Feb 9;84(3):345–357. doi: 10.1016/s0092-8674(00)81279-9. [DOI] [PubMed] [Google Scholar]
  15. Haas T. A., Plow E. F. Integrin-ligand interactions: a year in review. Curr Opin Cell Biol. 1994 Oct;6(5):656–662. doi: 10.1016/0955-0674(94)90091-4. [DOI] [PubMed] [Google Scholar]
  16. Hautanen A., Gailit J., Mann D. M., Ruoslahti E. Effects of modifications of the RGD sequence and its context on recognition by the fibronectin receptor. J Biol Chem. 1989 Jan 25;264(3):1437–1442. [PubMed] [Google Scholar]
  17. Huang T. F. What have snakes taught us about integrins? Cell Mol Life Sci. 1998 Jun;54(6):527–540. doi: 10.1007/s000180050181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huang T. F., Wu Y. J., Ouyang C. Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom. Biochim Biophys Acta. 1987 Sep 11;925(3):248–257. doi: 10.1016/0304-4165(87)90189-9. [DOI] [PubMed] [Google Scholar]
  19. Hynes R. O. Integrins: a family of cell surface receptors. Cell. 1987 Feb 27;48(4):549–554. doi: 10.1016/0092-8674(87)90233-9. [DOI] [PubMed] [Google Scholar]
  20. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  21. Liu C. Z., Wang Y. W., Shen M. C., Huang T. F. Analysis of human platelet glycoprotein IIb-IIIa by fluorescein isothiocyanate-conjugated disintegrins with flow cytometry. Thromb Haemost. 1994 Dec;72(6):919–925. [PubMed] [Google Scholar]
  22. Lu X., Rahman S., Kakkar V. V., Authi K. S. Substitutions of proline 42 to alanine and methionine 46 to asparagine around the RGD domain of the neurotoxin dendroaspin alter its preferential antagonism to that resembling the disintegrin elegantin. J Biol Chem. 1996 Jan 5;271(1):289–294. doi: 10.1074/jbc.271.1.289. [DOI] [PubMed] [Google Scholar]
  23. McLane M. A., Marcinkiewicz C., Vijay-Kumar S., Wierzbicka-Patynowski I., Niewiarowski S. Viper venom disintegrins and related molecules. Proc Soc Exp Biol Med. 1998 Nov;219(2):109–119. doi: 10.3181/00379727-219-44322. [DOI] [PubMed] [Google Scholar]
  24. Mustard J. F., Kinlough-Rathbone R. L., Packham M. A. Isolation of human platelets from plasma by centrifugation and washing. Methods Enzymol. 1989;169:3–11. doi: 10.1016/0076-6879(89)69045-3. [DOI] [PubMed] [Google Scholar]
  25. Parise L. V. Integrin alpha(IIb)beta(3) signaling in platelet adhesion and aggregation. Curr Opin Cell Biol. 1999 Oct;11(5):597–601. doi: 10.1016/s0955-0674(99)00018-6. [DOI] [PubMed] [Google Scholar]
  26. Pierschbacher M. D., Ruoslahti E. Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion. J Biol Chem. 1987 Dec 25;262(36):17294–17298. [PubMed] [Google Scholar]
  27. Rahman S., Aitken A., Flynn G., Formstone C., Savidge G. F. Modulation of RGD sequence motifs regulates disintegrin recognition of alphaIIb beta3 and alpha5 beta1 integrin complexes. Replacement of elegantin alanine-50 with proline, N-terminal to the RGD sequence, diminishes recognition of the alpha5 beta1 complex with restoration induced by Mn2+ cation. Biochem J. 1998 Oct 15;335(Pt 2):247–257. doi: 10.1042/bj3350247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rahman S., Flynn G., Aitken A., Patel Y., Hussain F., Lu X., Loftus J. C., French D., Wijelath E., Strand K. Differential recognition of snake venom proteins expressing specific Arg-Gly-Asp (RGD) sequence motifs by wild-type and variant integrin alphaIIbbeta3: further evidence for distinct sites of RGD ligand recognition exhibiting negative allostery. Biochem J. 2000 Feb 1;345(Pt 3):701–709. [PMC free article] [PubMed] [Google Scholar]
  29. Rahman S., Lu X., Kakkar V. V., Authi K. S. The integrin alpha IIb beta 3 contains distinct and interacting binding sites for snake-venom RGD (Arg-Gly-Asp) proteins. Evidence that the receptor-binding characteristics of snake-venom RGD proteins are related to the amino acid environment flanking the sequence RGD. Biochem J. 1995 Nov 15;312(Pt 1):223–232. doi: 10.1042/bj3120223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ruoslahti E., Pierschbacher M. D. Arg-Gly-Asp: a versatile cell recognition signal. Cell. 1986 Feb 28;44(4):517–518. doi: 10.1016/0092-8674(86)90259-x. [DOI] [PubMed] [Google Scholar]
  31. Scaloni A., Di Martino E., Miraglia N., Pelagalli A., Della Morte R., Staiano N., Pucci P. Amino acid sequence and molecular modelling of glycoprotein IIb-IIIa and fibronectin receptor iso-antagonists from Trimeresurus elegans venom. Biochem J. 1996 Nov 1;319(Pt 3):775–782. doi: 10.1042/bj3190775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Scarborough R. M., Naughton M. A., Teng W., Rose J. W., Phillips D. R., Nannizzi L., Arfsten A., Campbell A. M., Charo I. F. Design of potent and specific integrin antagonists. Peptide antagonists with high specificity for glycoprotein IIb-IIIa. J Biol Chem. 1993 Jan 15;268(2):1066–1073. [PubMed] [Google Scholar]
  33. Scarborough R. M., Rose J. W., Naughton M. A., Phillips D. R., Nannizzi L., Arfsten A., Campbell A. M., Charo I. F. Characterization of the integrin specificities of disintegrins isolated from American pit viper venoms. J Biol Chem. 1993 Jan 15;268(2):1058–1065. [PubMed] [Google Scholar]
  34. Shattil S. J., Ginsberg M. H., Brugge J. S. Adhesive signaling in platelets. Curr Opin Cell Biol. 1994 Oct;6(5):695–704. doi: 10.1016/0955-0674(94)90096-5. [DOI] [PubMed] [Google Scholar]
  35. Sutcliffe M. J., Jaseja M., Hyde E. I., Lu X., Williams J. A. Three-dimensional structure of the RGD-containing neurotoxin homologue dendroaspin. Nat Struct Biol. 1994 Nov;1(11):802–807. doi: 10.1038/nsb1194-802. [DOI] [PubMed] [Google Scholar]
  36. Tamkun J. W., DeSimone D. W., Fonda D., Patel R. S., Buck C., Horwitz A. F., Hynes R. O. Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin. Cell. 1986 Jul 18;46(2):271–282. doi: 10.1016/0092-8674(86)90744-0. [DOI] [PubMed] [Google Scholar]
  37. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES