Journal of Protein Chemistry

, Volume 13, Issue 1, pp 49–57 | Cite as

Covalent structure of botulinum neurotoxin type A: Location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains

  • Kerstin G. Kriegistein
  • Bibhuti R. DasGupta
  • Agnes H. Henschen
Article

Abstract

Botulinum neurotoxin Type A is synthesized byClostridium botulinum as a ∼150 kD single chain polypeptide. The posttranslational processing of the 1296 amino acid residue long gene product involves removal of the initiating methionine, formation of disulfide bridges, and limited proteolysis (nicking) by the bacterial protease(s). The mature dichain neurotoxin is made of a ∼50-kD light chain and a ∼100-kD heavy chain connected by a disulfide bridge. DNA derived amino acid sequencepredicted a total of 9 Cys residues (Binzet al., 1990,J. Biol. Chem.265, 9153–9158; Thompsonet al., 1990,Eur. J. Biochem.189, 73–81). Treatment of the dichain neurotoxin, dissolved in 6 M guanidine. HCl, with 4-vinylpyridine converted 5 Cys residues into S-pyridylethyl cysteine residues; but alkylation after mercaptolysis converted all 9 Cys residues in the S-pyridylethylated form. After confirming the predicted number of Cys residues by amino acid analysis, the positions of the 5 Cys residues carrying sulfhydryl groups and the 4 involved in disulfide bridges were determined by comparing the elution patterns in reversed-phase HPLC of the cyanogen bromide mixtures of the exclusively alkylated and the mercaptolyzed-alkylated neurotoxin. The chromatographically isolated components were identified by N-terminal amino acid sequence analysis. The HPLC patterns showed characteristic differences. The Cys residuespredicted in positions 133, 164, 790, 966, and 1059 were found in the sulfhydryl form; Cys 429 and 453 were found disulfide-bridged connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-bridge near the C-terminus in the heavy chain. Ten amino acid residues, Thr 438-Lys 447,predicted to be present in the single chain neurotoxin were not found in the dichain neurotoxin. Nicking of single-chain neurotoxin by the protease(s) endogenous to the bacteria therefore appears to excise these 10 amino acid residues from the nicking region which leaves Lys 437 as the C-terminus of the light chain and Ala 448 as the N-terminus of the heavy chain. The N-terminal Pro 1 and C-terminal Leu 1295,predicted from the nucleotide sequence, remain conserved after nicking. Residues Pro 1-Lys 437 and Ala 448-Leu 1295 constitute the light and heavy chains, respectively. The C-termini were determined by isolation of short C-terminal peptide fragments and subsequent sequence analysis by Edman degradation. About 20% of the amino acid sequence predicted from DNA analysis was confirmed in these studies by protein-chemical methods.

Key words

Botulinum neurotoxin CNBr fragmentation HPLC separation sulfhydryl disulfide C-termini 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Barinaga, M. (1993).Science 260, 487–489.PubMedGoogle Scholar
  2. Betley, M., Somers, E., and DasGupta, B. R. (1989).Biochem. Biophys. Res. Commun. 162, 1388–1395.PubMedGoogle Scholar
  3. Binz, T., Kurazono, H., Popoff, M. R., Eklund, M. W., Sakaguchi, G., Kozaki, S., Krieglstein, K., Henschen, A., Gill, D. M., and Niemann, H. (1990b).Nucl. Acids Res. 18, 5556.PubMedGoogle Scholar
  4. Binz, T., Kurazono, H., Wille, M., Frevert, J., Wernars, K., and Niemann, H. (1990a).J. Biol. Chem. 265, 9153–9158.PubMedGoogle Scholar
  5. DasGupta, B. R. (1990).J. Physiol. (Paris) 84, 220–228.Google Scholar
  6. DasGupta, B. R., and Dekleva, M. L. (1990).Biochimie 72, 661–664.PubMedGoogle Scholar
  7. DasGupta, B. R., and Foley, J. (1989).Biochimie 71, 1193–1200.PubMedGoogle Scholar
  8. DasGupta, B. R., and Sathyamoorthy, V. (1984).Toxincon 22, 415–424.Google Scholar
  9. DasGupta, B. R., and Sugiyama, H. (1972).Biochem. Biophys. Res. Commun. 48, 108–112.PubMedGoogle Scholar
  10. DasGupta, B. R., and Sugiyama, H. (1978).Abstr. Am. Soc. Microbiol. p. 25.Google Scholar
  11. DeKleva, M. L., and DasGupta, B. R. (1989).Biochem. Biophys. Res. Commun. 162, 767–772.PubMedGoogle Scholar
  12. East, A. K., Richardson, P. T., Allaway, D., Collins, M. D., Roberts, T. A., and Thompson, D. E. (1992).FEMS Microbiol. Lett. 96, 225–230.Google Scholar
  13. Edman, P., and Henschen, A. (1975). InProtein Sequence Determination (Needleman, S. B., ed.), 2nd ed., Springer-Verlag, Berlin, pp. 232–279.Google Scholar
  14. Gimenez, J. A., and DasGupta, B. R. (1993).J. Protein Chem. 12, 349–361.Google Scholar
  15. Hauser, D., Eklund, M. W., Kurazono, H., Binz, T., Neimann, H., Gill, D. M., Boquet, P., and Popoff, M. R. (1990).Nucl. Acids Res. 18, 4924.PubMedGoogle Scholar
  16. Henschen, A. (1986). InAdvanced Methods in Protein Microsequence Analysis (Wittmann-Liebold, B., Salnikow, J., and Erdmann, V. A., eds.), Springer-Verlag, Berlin, pp. 244–255.Google Scholar
  17. Jankovic, J., and Brin, M. F. (1991).New Eng. J. Med. 324, 1186–1194.PubMedGoogle Scholar
  18. Krieglstein, K., Henschen, A., Weller, U., and Habermann, E. (1990).Eur. J. Biochem. 188, 39–45.PubMedGoogle Scholar
  19. Kurazono, H., Mochida, S., Binz, T., Eisel, U., Quanz, M., Grebenstein, O., Wernars, K., Poulains, B., Tauc, L., and Niemann, H. (1922).J. Biol. Chem. 267, 14721–14729.Google Scholar
  20. Lottspeiich, F., and Henschen, A. (1985). InHigh-performance Liquid Chormatography in Biochemistry (Henschen, A., Hupe, K. P., Lottspeich, F., and Boelter, W., eds.), Verlag Chemie, Weinheim, pp. 139–216.Google Scholar
  21. Niemann, H. (1991). InSourcebook of Bacterial Protein Toxins (Alouf, J., and Freer, J., eds.), Academic Press, New York, pp. 303–348.Google Scholar
  22. Poulet, S., Hauser, D., Quanz, M., Niemann, H., and Popoff, M. R. (1992).Biochem. Biophys. Res. Commun. 183, 107–113.PubMedGoogle Scholar
  23. Sathyammorty, V., DasGupta, B. R., Foley, J., and Niece, R. L., (1988).Arch. Biochem. Biophys. 266, 142–151.PubMedGoogle Scholar
  24. Sathyamoorthy, V., and DasGupta, B. R. (1985).J. Biol. Chem. 260, 10461–10466.PubMedGoogle Scholar
  25. Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., de Laureto, P. P., DasGupta, B. R., and Montecucco, C. (1992).Nature 359, 832–834.PubMedGoogle Scholar
  26. Schmidt, J. J., Sathyamoorthy, V., and DasGupta, B. R. (1984).Biochem. Biophys. Res. Commun. 119, 900–904.PubMedGoogle Scholar
  27. Shone, C. C., Hembleton, P., and Melling, J. (1985).Eur. J. Biochem. 151, 75–82.PubMedGoogle Scholar
  28. Stevens, R. C., Evenson, J. L., Tepp, W., and DasGupta, B. R. (1991).J. Mol. Biol. 222, 877–880.PubMedGoogle Scholar
  29. Thompson, D. E., Brehm, J. K., Oultram, J. D., Sweingfield, T.-J., Shone, C. C., Atkinson, T., Melling, J., and Minton, N. P. (1990).Eur. J. Biochem. 189, 73–81.PubMedGoogle Scholar
  30. Whelan, S. M., Elmore, M. J., Bodsworth, N. J., Atkinson, T., and Minton, N. P. (1992b).Eur. J. Biochem. 204, 657–667.PubMedGoogle Scholar
  31. Whelan, S. M., Elmore, M. J., Bodsworth, N. J., Brehm, J. K., Atkinson, T., and Minton, N. P. (1992a).Appl. Environ. Microbiol. 58, 2345–2354.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1994

Authors and Affiliations

  • Kerstin G. Kriegistein
    • 1
  • Bibhuti R. DasGupta
    • 2
  • Agnes H. Henschen
    • 1
  1. 1.Department of Molecular Biology and BiochemistryUniversity of CaliforniaIrvine
  2. 2.Department of Food Microbiology and ToxicologyUniversity of WisconsinMadison

Personalised recommendations