• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of iaiPermissionsJournals.ASM.orgJournalIAI ArticleJournal InfoAuthorsReviewers
Infect Immun. May 1997; 65(5): 1626–1630.
PMCID: PMC175186

Antibody mapping to domains of botulinum neurotoxin serotype A in the complexed and uncomplexed forms.

Abstract

The domain organization of the botulinum neurotoxin serotype A was studied by using antibody mapping of 44 monoclonal single-chain variable fragments. The analysis was carried out on (i) the individual domains of botulinum neurotoxin holotoxin (binding, translocation, and catalytic), (ii) botulinum neurotoxin holotoxin, (iii) the botulinum neurotoxin holotoxin in complex with the nontoxic portion, and (iv) botulinum neurotoxin holotoxin and nontoxic portion of the complex recombined in vitro. All 44 antibodies mapped to individual domains of botulinum neurotoxin. Forty of the 44 single-chain variable fragments bound the botulinum neurotoxin holotoxin relative to the isolated domains, suggesting that 4 epitopes are covered when the individual domains are in the holotoxin form. Only 20 of the antibodies showed a positive reaction to the toxin while in complex with the nontoxic portion. All of the covered epitopes were mapped to the binding domain of botulinum neurotoxin, which suggested that the binding domain is in direct contact with the nontoxic portion in the complex. Based on the antibody mapping to the different domains of the botulinum neurotoxin holotoxin and the entire complex, a model of the botulinum neurotoxin complex is proposed.

Full Text

The Full Text of this article is available as a PDF (189K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Blasi J, Chapman ER, Link E, Binz T, Yamasaki S, De Camilli P, Südhof TC, Niemann H, Jahn R. Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25. Nature. 1993 Sep 9;365(6442):160–163. [PubMed]
  • Blaustein RO, Germann WJ, Finkelstein A, DasGupta BR. The N-terminal half of the heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayers. FEBS Lett. 1987 Dec 21;226(1):115–120. [PubMed]
  • DasGupta BR, Boroff DA. Separation of toxin and hemagglutinin from crystalline toxin of Clostridium botulinum type A by anion exchange chromatography and determination of their dimensions by gel filtration. J Biol Chem. 1968 Mar 10;243(5):1065–1072. [PubMed]
  • Dasgupta BR, Boroff DA, Rothstein E. Chromatographic fractionation of the crystalline toxin of Clostridium botulinum type A. Biochem Biophys Res Commun. 1966 Mar 22;22(6):750–756. [PubMed]
  • DasGupta BR, Sathyamoorthy V. Purification and amino acid composition of type A botulinum neurotoxin. Toxicon. 1984;22(3):415–424. [PubMed]
  • De Bellis D, Schwartz I. Regulated expression of foreign genes fused to lac: control by glucose levels in growth medium. Nucleic Acids Res. 1990 Mar 11;18(5):1311–1311. [PMC free article] [PubMed]
  • Ekong TA, McLellan K, Sesardic D. Immunological detection of Clostridium botulinum toxin type A in therapeutic preparations. J Immunol Methods. 1995 Mar 27;180(2):181–191. [PubMed]
  • Fling SP, Gregerson DS. Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea. Anal Biochem. 1986 May 15;155(1):83–88. [PubMed]
  • Hayashi T, McMahon H, Yamasaki S, Binz T, Hata Y, Südhof TC, Niemann H. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 1994 Nov 1;13(21):5051–5061. [PMC free article] [PubMed]
  • Hoogenboom HR, Griffiths AD, Johnson KS, Chiswell DJ, Hudson P, Winter G. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 1991 Aug 11;19(15):4133–4137. [PMC free article] [PubMed]
  • Knox J, Brown WP, Spero L. The role of sulfhydryl groups in the activity of type A botulinum toxin. Biochim Biophys Acta. 1970 Aug 21;214(2):350–354. [PubMed]
  • Kozaki S, Kamata Y, Nagai T, Ogasawara J, Sakaguchi G. The use of monoclonal antibodies to analyze the structure of Clostridium botulinum type E derivative toxin. Infect Immun. 1986 Jun;52(3):786–791. [PMC free article] [PubMed]
  • Kozaki S, Miki A, Kamata Y, Ogasawara J, Sakaguchi G. Immunological characterization of papain-induced fragments of Clostridium botulinum type A neurotoxin and interaction of the fragments with brain synaptosomes. Infect Immun. 1989 Sep;57(9):2634–2639. [PMC free article] [PubMed]
  • Kozaki S, Nakaue S, Kamata Y. Immunological characterization of the neurotoxin produced by Clostridium botulinum type A associated with infant botulism in Japan. Microbiol Immunol. 1995;39(10):767–774. [PubMed]
  • Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol. 1991 Dec 5;222(3):581–597. [PubMed]
  • Munro S, Pelham HR. An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell. 1986 Jul 18;46(2):291–300. [PubMed]
  • Sathyamoorthy V, Dasgupta BR, Foley J, Niece RL. Botulinum neurotoxin type A: cleavage of the heavy chain into two halves and their partial sequences. Arch Biochem Biophys. 1988 Oct;266(1):142–151. [PubMed]
  • Schier R, Balint RF, McCall A, Apell G, Larrick JW, Marks JD. Identification of functional and structural amino-acid residues by parsimonious mutagenesis. Gene. 1996 Mar 9;169(2):147–155. [PubMed]
  • Schier R, Bye J, Apell G, McCall A, Adams GP, Malmqvist M, Weiner LM, Marks JD. Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection. J Mol Biol. 1996 Jan 12;255(1):28–43. [PubMed]
  • Shone CC, Hambleton P, Melling J. Inactivation of Clostridium botulinum type A neurotoxin by trypsin and purification of two tryptic fragments. Proteolytic action near the COOH-terminus of the heavy subunit destroys toxin-binding activity. Eur J Biochem. 1985 Aug 15;151(1):75–82. [PubMed]
  • Simpson LL. Kinetic studies on the interaction between botulinum toxin type A and the cholinergic neuromuscular junction. J Pharmacol Exp Ther. 1980 Jan;212(1):16–21. [PubMed]
  • Sugii S, Ohishi I, Sakaguchi G. Correlation between oral toxicity and in vitro stability of Clostridium botulinum type A and B toxins of different molecular sizes. Infect Immun. 1977 Jun;16(3):910–914. [PMC free article] [PubMed]
  • Sugii S, Ohishi I, Sakaguchi G. Intestinal absorption of botulinum toxins of different molecular sizes in rats. Infect Immun. 1977 Sep;17(3):491–496. [PMC free article] [PubMed]
  • Sugiyama H. Clostridium botulinum neurotoxin. Microbiol Rev. 1980 Sep;44(3):419–448. [PMC free article] [PubMed]
  • Sugiyama H, Oishi I, Dasgupta BR. Evaluation of type A botulinal toxin assays that use antitoxin to crystalline toxin. Appl Microbiol. 1974 Feb;27(2):333–336. [PMC free article] [PubMed]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...