Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Jun 15;372(Pt 3):725–734. doi: 10.1042/BJ20021739

Snake venom disintegrins: novel dimeric disintegrins and structural diversification by disulphide bond engineering.

Juan J Calvete 1, M Paz Moreno-Murciano 1, R David G Theakston 1, Dariusz G Kisiel 1, Cezary Marcinkiewicz 1
PMCID: PMC1223455  PMID: 12667142

Abstract

We report the isolation and amino acid sequences of six novel dimeric disintegrins from the venoms of Vipera lebetina obtusa (VLO), V. berus (VB), V. ammodytes (VA), Echis ocellatus (EO) and Echis multisquamatus (EMS). Disintegrins VLO4, VB7, VA6 and EO4 displayed the RGD motif and inhibited the adhesion of K562 cells, expressing the integrin alpha5beta1 to immobilized fibronectin. A second group of dimeric disintegrins (VLO5 and EO5) had MLD and VGD motifs in their subunits and blocked the adhesion of the alpha4beta1 integrin to vascular cell adhesion molecule 1 with high selectivity. On the other hand, disintegrin EMS11 inhibited both alpha5beta1 and alpha4beta1 integrins with almost the same degree of specificity. Comparison of the amino acid sequences of the dimeric disintegrins with those of other disintegrins by multiple-sequence alignment and phylogenetic analysis, in conjunction with current biochemical and genetic data, supports the view that the different disintegrin subfamilies evolved from a common ADAM (a disintegrin and metalloproteinase-like) scaffold and that structural diversification occurred through disulphide bond engineering.

Full Text

The Full Text of this article is available as a PDF (498.8 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. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bauer M., Sun Y., Degenhardt C., Kozikowski B. Assignment of all four disulfide bridges in echistatin. J Protein Chem. 1993 Dec;12(6):759–764. doi: 10.1007/BF01024934. [DOI] [PubMed] [Google Scholar]
  4. Calvete J. J., Jürgens M., Marcinkiewicz C., Romero A., Schrader M., Niewiarowski S. Disulphide-bond pattern and molecular modelling of the dimeric disintegrin EMF-10, a potent and selective integrin alpha5beta1 antagonist from Eristocophis macmahoni venom. Biochem J. 2000 Feb 1;345(Pt 3):573–581. [PMC free article] [PubMed] [Google Scholar]
  5. Calvete J. J., Moreno-Murciano M. P., Sanz L., Jürgens M., Schrader M., Raida M., Benjamin D. C., Fox J. W. The disulfide bond pattern of catrocollastatin C, a disintegrin-like/cysteine-rich protein isolated from Crotalus atrox venom. Protein Sci. 2000 Jul;9(7):1365–1373. doi: 10.1110/ps.9.7.1365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Calvete J. J., Schrader M., Raida M., McLane M. A., Romero A., Niewiarowski S. The disulphide bond pattern of bitistatin, a disintegrin isolated from the venom of the viper Bitis arietans. FEBS Lett. 1997 Oct 20;416(2):197–202. doi: 10.1016/s0014-5793(97)01203-9. [DOI] [PubMed] [Google Scholar]
  7. Calvete J. J., Schäfer W., Soszka T., Lu W. Q., Cook J. J., Jameson B. A., Niewiarowski S. Identification of the disulfide bond pattern in albolabrin, an RGD-containing peptide from the venom of Trimeresurus albolabris: significance for the expression of platelet aggregation inhibitory activity. Biochemistry. 1991 May 28;30(21):5225–5229. doi: 10.1021/bi00235a016. [DOI] [PubMed] [Google Scholar]
  8. Calvete J. J., Wang Y., Mann K., Schäfer W., Niewiarowski S., Stewart G. J. The disulfide bridge pattern of snake venom disintegrins, flavoridin and echistatin. FEBS Lett. 1992 Sep 14;309(3):316–320. doi: 10.1016/0014-5793(92)80797-k. [DOI] [PubMed] [Google Scholar]
  9. Calvete Juan J., Fox Jay W., Agelan Alexis, Niewiarowski Stefan, Marcinkiewicz Cezary. The presence of the WGD motif in CC8 heterodimeric disintegrin increases its inhibitory effect on alphaII(b)beta3, alpha(v)beta3, and alpha5beta1 integrins. Biochemistry. 2002 Feb 12;41(6):2014–2021. doi: 10.1021/bi015627o. [DOI] [PubMed] [Google Scholar]
  10. Duda T. F., Jr, Palumbi S. R. Molecular genetics of ecological diversification: duplication and rapid evolution of toxin genes of the venomous gastropod Conus. Proc Natl Acad Sci U S A. 1999 Jun 8;96(12):6820–6823. doi: 10.1073/pnas.96.12.6820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gasmi A., Srairi N., Guermazi S., Dekhil H., Dkhil H., Karoui H., El Ayeb M. Amino acid structure and characterization of a heterodimeric disintegrin from Vipera lebetina venom. Biochim Biophys Acta. 2001 May 5;1547(1):51–56. doi: 10.1016/s0167-4838(01)00168-6. [DOI] [PubMed] [Google Scholar]
  12. Hong Sung-Yu, Koh You-Seok, Chung Kwang-Hoe, Kim Doo-Sik. Snake venom disintegrin, saxatilin, inhibits platelet aggregation, human umbilical vein endothelial cell proliferation, and smooth muscle cell migration. Thromb Res. 2002 Jan 1;105(1):79–86. doi: 10.1016/s0049-3848(01)00416-9. [DOI] [PubMed] [Google Scholar]
  13. Huang T. F., Holt J. C., Lukasiewicz H., Niewiarowski S. Trigramin. A low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein IIb-IIIa complex. J Biol Chem. 1987 Nov 25;262(33):16157–16163. [PubMed] [Google Scholar]
  14. Ito M., Hamako J., Sakurai Y., Matsumoto M., Fujimura Y., Suzuki M., Hashimoto K., Titani K., Matsui T. Complete amino acid sequence of kaouthiagin, a novel cobra venom metalloproteinase with two disintegrin-like sequences. Biochemistry. 2001 Apr 10;40(14):4503–4511. doi: 10.1021/bi0022700. [DOI] [PubMed] [Google Scholar]
  15. Jia L. G., Shimokawa K., Bjarnason J. B., Fox J. W. Snake venom metalloproteinases: structure, function and relationship to the ADAMs family of proteins. Toxicon. 1996 Nov-Dec;34(11-12):1269–1276. doi: 10.1016/s0041-0101(96)00108-0. [DOI] [PubMed] [Google Scholar]
  16. Kini R. M., Evans H. J. Structural domains in venom proteins: evidence that metalloproteinases and nonenzymatic platelet aggregation inhibitors (disintegrins) from snake venoms are derived by proteolysis from a common precursor. Toxicon. 1992 Mar;30(3):265–293. doi: 10.1016/0041-0101(92)90869-7. [DOI] [PubMed] [Google Scholar]
  17. Klaus W., Broger C., Gerber P., Senn H. Determination of the disulphide bonding pattern in proteins by local and global analysis of nuclear magnetic resonance data. Application to flavoridin. J Mol Biol. 1993 Aug 5;232(3):897–906. doi: 10.1006/jmbi.1993.1438. [DOI] [PubMed] [Google Scholar]
  18. Kumar S., Tamura K., Jakobsen I. B., Nei M. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics. 2001 Dec;17(12):1244–1245. doi: 10.1093/bioinformatics/17.12.1244. [DOI] [PubMed] [Google Scholar]
  19. Marcinkiewicz C., Calvete J. J., Marcinkiewicz M. M., Raida M., Vijay-Kumar S., Huang Z., Lobb R. R., Niewiarowski S. EC3, a novel heterodimeric disintegrin from Echis carinatus venom, inhibits alpha4 and alpha5 integrins in an RGD-independent manner. J Biol Chem. 1999 Apr 30;274(18):12468–12473. doi: 10.1074/jbc.274.18.12468. [DOI] [PubMed] [Google Scholar]
  20. Marcinkiewicz C., Calvete J. J., Vijay-Kumar S., Marcinkiewicz M. M., Raida M., Schick P., Lobb R. R., Niewiarowski S. Structural and functional characterization of EMF10, a heterodimeric disintegrin from Eristocophis macmahoni venom that selectively inhibits alpha 5 beta 1 integrin. Biochemistry. 1999 Oct 5;38(40):13302–13309. doi: 10.1021/bi9906930. [DOI] [PubMed] [Google Scholar]
  21. Marcinkiewicz C., Taooka Y., Yokosaki Y., Calvete J. J., Marcinkiewicz M. M., Lobb R. R., Niewiarowski S., Sheppard D. Inhibitory effects of MLDG-containing heterodimeric disintegrins reveal distinct structural requirements for interaction of the integrin alpha 9beta 1 with VCAM-1, tenascin-C, and osteopontin. J Biol Chem. 2000 Oct 13;275(41):31930–31937. doi: 10.1074/jbc.M003209200. [DOI] [PubMed] [Google Scholar]
  22. Marcinkiewicz C., Vijay-Kumar S., McLane M. A., Niewiarowski S. Significance of RGD loop and C-terminal domain of echistatin for recognition of alphaIIb beta3 and alpha(v) beta3 integrins and expression of ligand-induced binding site. Blood. 1997 Aug 15;90(4):1565–1575. [PubMed] [Google Scholar]
  23. Markland F. S. Snake venoms and the hemostatic system. Toxicon. 1998 Dec;36(12):1749–1800. doi: 10.1016/s0041-0101(98)00126-3. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. McLane M. A., Vijay-Kumar S., Marcinkiewicz C., Calvete J. J., Niewiarowski S. Importance of the structure of the RGD-containing loop in the disintegrins echistatin and eristostatin for recognition of alpha IIb beta 3 and alpha v beta 3 integrins. FEBS Lett. 1996 Aug 5;391(1-2):139–143. doi: 10.1016/0014-5793(96)00716-8. [DOI] [PubMed] [Google Scholar]
  26. Moura-da-Silva A. M., Theakston R. D., Crampton J. M. Evolution of disintegrin cysteine-rich and mammalian matrix-degrading metalloproteinases: gene duplication and divergence of a common ancestor rather than convergent evolution. J Mol Evol. 1996 Sep;43(3):263–269. doi: 10.1007/BF02338834. [DOI] [PubMed] [Google Scholar]
  27. Neeper M. P., Jacobson M. A. Sequence of a cDNA encoding the platelet aggregation inhibitor trigramin. Nucleic Acids Res. 1990 Jul 25;18(14):4255–4255. doi: 10.1093/nar/18.14.4255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nikai T., Taniguchi K., Komori Y., Masuda K., Fox J. W., Sugihara H. Primary structure and functional characterization of bilitoxin-1, a novel dimeric P-II snake venom metalloproteinase from Agkistrodon bilineatus venom. Arch Biochem Biophys. 2000 Jun 1;378(1):6–15. doi: 10.1006/abbi.2000.1795. [DOI] [PubMed] [Google Scholar]
  29. Okuda D., Morita T. Purification and characterization of a new RGD/KGD-containing dimeric disintegrin, piscivostatin, from the venom of Agkistrodon piscivorus piscivorus: the unique effect of piscivostatin on platelet aggregation. J Biochem. 2001 Sep;130(3):407–415. doi: 10.1093/oxfordjournals.jbchem.a003000. [DOI] [PubMed] [Google Scholar]
  30. Okuda D., Nozaki C., Sekiya F., Morita T. Comparative biochemistry of disintegrins isolated from snake venom: consideration of the taxonomy and geographical distribution of snakes in the genus Echis. J Biochem. 2001 Apr;129(4):615–620. doi: 10.1093/oxfordjournals.jbchem.a002898. [DOI] [PubMed] [Google Scholar]
  31. Oshikawa K., Terada S. Ussuristatin 2, a novel KGD-bearing disintegrin from Agkistrodon ussuriensis venom. J Biochem. 1999 Jan;125(1):31–35. doi: 10.1093/oxfordjournals.jbchem.a022264. [DOI] [PubMed] [Google Scholar]
  32. Park D., Kang I., Kim H., Chung K., Kim D. S., Yun Y. Cloning and characterization of novel disintegrins from Agkistrodon halys venom. Mol Cells. 1998 Oct 31;8(5):578–584. [PubMed] [Google Scholar]
  33. Park D., Kang I., Kim H., Chung K., Kim D. S., Yun Y. Cloning and characterization of novel disintegrins from Agkistrodon halys venom. Mol Cells. 1998 Oct 31;8(5):578–584. [PubMed] [Google Scholar]
  34. Saudek V., Atkinson R. A., Pelton J. T. Three-dimensional structure of echistatin, the smallest active RGD protein. Biochemistry. 1991 Jul 30;30(30):7369–7372. doi: 10.1021/bi00244a003. [DOI] [PubMed] [Google Scholar]
  35. Scarborough R. M., Rose J. W., Hsu M. A., Phillips D. R., Fried V. A., Campbell A. M., Nannizzi L., Charo I. F. Barbourin. A GPIIb-IIIa-specific integrin antagonist from the venom of Sistrurus m. barbouri. J Biol Chem. 1991 May 25;266(15):9359–9362. [PubMed] [Google Scholar]
  36. Senn H., Klaus W. The nuclear magnetic resonance solution structure of flavoridin, an antagonist of the platelet GP IIb-IIIa receptor. J Mol Biol. 1993 Aug 5;232(3):907–925. doi: 10.1006/jmbi.1993.1439. [DOI] [PubMed] [Google Scholar]
  37. Shimokawa K., Jia L. G., Shannon J. D., Fox J. W. Isolation, sequence analysis, and biological activity of atrolysin E/D, the non-RGD disintegrin domain from Crotalus atrox venom. Arch Biochem Biophys. 1998 Jun 15;354(2):239–246. doi: 10.1006/abbi.1998.0698. [DOI] [PubMed] [Google Scholar]
  38. Shimokawa K., Shannon J. D., Jia L. G., Fox J. W. Sequence and biological activity of catrocollastatin-C: a disintegrin-like/cysteine-rich two-domain protein from Crotalus atrox venom. Arch Biochem Biophys. 1997 Jul 1;343(1):35–43. doi: 10.1006/abbi.1997.0133. [DOI] [PubMed] [Google Scholar]
  39. Smith J. Bryan, Theakston R. David G., Coelho Ana Lucia J., Barja-Fidalgo Christina, Calvete Juan J., Marcinkiewicz Cezary. Characterization of a monomeric disintegrin, ocellatusin, present in the venom of the Nigerian carpet viper, Echis ocellatus. FEBS Lett. 2002 Feb 13;512(1-3):111–115. doi: 10.1016/s0014-5793(02)02233-0. [DOI] [PubMed] [Google Scholar]
  40. Smith K. J., Jaseja M., Lu X., Williams J. A., Hyde E. I., Trayer I. P. Three-dimensional structure of the RGD-containing snake toxin albolabrin in solution, based on 1H NMR spectroscopy and simulated annealing calculations. Int J Pept Protein Res. 1996 Sep;48(3):220–228. doi: 10.1111/j.1399-3011.1996.tb00835.x. [DOI] [PubMed] [Google Scholar]
  41. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Trabesinger-Ruef N., Jermann T., Zankel T., Durrant B., Frank G., Benner S. A. Pseudogenes in ribonuclease evolution: a source of new biomacromolecular function? FEBS Lett. 1996 Mar 18;382(3):319–322. doi: 10.1016/0014-5793(96)00191-3. [DOI] [PubMed] [Google Scholar]
  43. Trikha M., De Clerck Y. A., Markland F. S. Contortrostatin, a snake venom disintegrin, inhibits beta 1 integrin-mediated human metastatic melanoma cell adhesion and blocks experimental metastasis. Cancer Res. 1994 Sep 15;54(18):4993–4998. [PubMed] [Google Scholar]
  44. Wu W. B., Chang S. C., Liau M. Y., Huang T. F. Purification, molecular cloning and mechanism of action of graminelysin I, a snake-venom-derived metalloproteinase that induces apoptosis of human endothelial cells. Biochem J. 2001 Aug 1;357(Pt 3):719–728. doi: 10.1042/0264-6021:3570719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Xiong Jian-Ping, Stehle Thilo, Zhang Rongguang, Joachimiak Andrzej, Frech Matthias, Goodman Simon L., Arnaout M. Amin. Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand. Science. 2002 Mar 7;296(5565):151–155. doi: 10.1126/science.1069040. [DOI] [PubMed] [Google Scholar]
  46. Zhou Q., Hu P., Ritter M. R., Swenson S. D., Argounova S., Epstein A. L., Markland F. S. Molecular cloning and functional expression of contortrostatin, a homodimeric disintegrin from southern copperhead snake venom. Arch Biochem Biophys. 2000 Mar 15;375(2):278–288. doi: 10.1006/abbi.1999.1682. [DOI] [PubMed] [Google Scholar]
  47. Zhou Q., Smith J. B., Grossman M. H. Molecular cloning and expression of catrocollastatin, a snake-venom protein from Crotalus atrox (western diamondback rattlesnake) which inhibits platelet adhesion to collagen. Biochem J. 1995 Apr 15;307(Pt 2):411–417. doi: 10.1042/bj3070411. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES