1887

Microbiology Society logo

Volume 138, Issue 10

Targeting of specific domains of diphtheria toxin by site-directed antibodies Free

Abstract

Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx on fragment B, selected on the basis of forming a predicted surface loop; and DTx on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominately against a narrow region within the peptide, consisting of only 5–6 aa residues. The predicted location of the peptide and their epitopes was confirmed by inspection of the X-ray crystallographic structure of DTx. Antibodies to peptides were selective for the toxin, one binding to DTx some 5–60-fold better than to diphtheria toxoid, presumably reflecting variability caused by toxoid preparation at this epitope. None of the antisera produced protected against DTx challenge in the guinea pig intradermal test . Although the availability of site-specific antibodies that recognize neutralizing epitopes would be very valuable, antibodies such as those described here should prove extremely useful in the structure-function analysis of DTx.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Society for General Microbiology, 1992
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-138-10-2197
1992-10-01
2024-05-12
Loading full text...

Full text loading...

/deliver/fulltext/micro/138/10/mic-138-10-2197.html?itemId=/content/journal/micro/10.1099/00221287-138-10-2197&mimeType=html&fmt=ahah

References

  1. Audibert F., , Olivet M., , Chedid L., , Alouf J. E., , Boquet P., , Rivaille P., & Siffert O. 1981; Active antitoxic imMunization by a diphtheria toxin synthetic oligopeptide. Nature, London 289:593–594
     [Google Scholar]
  2. Audibert F., , Jolivet M., , Chedid L., , Arnon R., & Sela M. 1982; Successful imMunization with a totally synthetic diphtheria vaccine. Proceedings of the National Academy of Sciences of the United States of America 19, 5042–5046
     [Google Scholar]
  3. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 12, 248–254
     [Google Scholar]
  4. Choe S., , Bennett M. J., , Furn G., , Curmi P. M. G., , Kantardjieff K. A., , Collier R. J., & Eisenberg D. 1992; The crystal structure of diphtheria toxin. Nature, London 357:216–222
     [Google Scholar]
  5. Cieplak W., , Gaudin H. M., & Eidels L. 1987; Diphtheria toxin receptor. Identification of specific diphtheria toxin-binding proteins on the surface of Vero and Bs-C-1 cells. Journal of Biological Chemistry 262:13246–13253
     [Google Scholar]
  6. Collier R. J., & Pappenheimer A. M. 1964; Studies on the mode of action of diphtheria toxin. II Effect of toxin on amino acid incorporation in cell-free systems. Journal of Experimental Medicine 120:1019–1039
     [Google Scholar]
  7. Devereux J., , Haeberli P., & Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 24:387–395
     [Google Scholar]
  8. Edwards R. J., , Singleton A. M., , Boobis A., & Davies D.S. 1989; Cross-reaction of antibodies to coupling groups used in the, production of anti-peptide antibodies. Journal of immunological Methods 117:215–220
     [Google Scholar]
  9. Edwards R. J., , Murray B. P., & Boobis A. R. 1991; Anti-peptide antibodies in studies of cytochrome P450IA. Methods in Enzymology 206:220–233
     [Google Scholar]
  10. Ellman G. L. 1959; Tissue sulfhydryl groups. Archives of Biochemis-try and Biophysics 82:70–77
     [Google Scholar]
  11. Geysen H. M., , Rodda S. J., , Mason T. J., , Tribbick G., & Schoofs P. G. 1987; Strategies for epitope analysis using peptide synthesis. Journal of immunological Methods 102:259–274
     [Google Scholar]
  12. Greenfield L., , Bjorn M. J., , Horn G., , Fong D., , Buck G. A., , Collier R. J., & Kaplan D. A. 1983; Nucleotide sequence of the structural gene for diphtheria toxin carried by corynebacteriophage β . Proceedings of the National AcademyofSciencesofthe United States of America 806853–6857
     [Google Scholar]
  13. Honjo T., , Nishizuka Y., , Hayaishi O., & Kato I. 1968; Diphtheria toxin-dependent adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis. Journal of Biological Chemistry 243: 3553-3555.
    [Google Scholar]
  14. Jones B. N., , Paabo S., & Stein S. 1981; Amino acid analysis and enzymatic sequence determination of peptides by an improved o- phthaldialdehyde precolumn labelling procedure. Journal of Liquid Chromatography 4:565–586
     [Google Scholar]
  15. Mekada E., , Okada Y., & Uchida T. 1988; Identification of diphtheria toxin receptor and a nonproteinous diphtheria toxin-binding molecule in Vero cell membrane. Journal of Cell Biology 107:511–519
     [Google Scholar]
  16. Pappenheimer A. M. 1977; Diphtheria toxin. Annual Review in Biochemistry 46:69–94
     [Google Scholar]
  17. Perera V. Y., & Corbel M. J. 1990; Human antibody response to fragments A and B of diphtheria toxin and a synthetic peptide of amino acid residues 141-157 of fragment A. Epidemiology and Infection 105:457–468
     [Google Scholar]
  18. Rolf J.M., , Gaudin H. M., & Eidels L. 1990; Localization of the diphtheria toxin receptor-binding domain to the carboxyl-terminal M r ≈ 6000 region of the toxin. Journal of Biological Chemistry 265:7331–7337
     [Google Scholar]
  19. Towbin H., , Staehelin T., & Gordon J. 1979; Electrophoretic transfer from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America 16, 4350–4354
     [Google Scholar]
  20. Tweten R. K., , Barbieri J. T., & Collier R. J. 1985; Effect of substituting aspartic acid for glutamic acid 148 on ADP-ribosyltrans-ferase activity. Journal of Biological Chemistry 260:10392–10394
     [Google Scholar]
  21. Uchida T., , Yamaizumi M., & Okada Y. 1977; Reassembled HVJ (Sendai virus) envelopes containing non-toxic mutant proteins of diphtheria toxin show toxicity to mouse L cell. Nature, London 266:839–840
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-138-10-2197
Loading
Targeting of specific domains of diphtheria toxin by site-directed antibodies
Microbiology 138, 2197 (1992); https://doi.org/10.1099/00221287-138-10-2197
/content/journal/micro/10.1099/00221287-138-10-2197
/content/journal/micro/10.1099/00221287-138-10-2197
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error