Abstract

Botulinum neurotoxin (NT) causes the neuroparalytic disease botulism generally following ingestion of food in which the ubiquitous spore forming bacteria Clostridium botulinum multiplied and produced the NT. Infant botulism, toxico-infection in 3-35 week old humans, results from ingestion of spores, not the preformed NT. Toxico-infection in adults is now known. Non- botulinum species C. butyricum and C. barati have caused human botulism. The immunologically distinct seven NT serotypes block release of the neurotransmitter acetylcholine (ACh) at neuromuscular junctions inducing flaccid paralysis. This property of the NT is being exploited to correct spastic muscle conditions such as strabismus (crossed eyes), blepharospasm hemifacial spasm etc. Very little is known about structure of the NT and its mode of action, other than it binds to the nerve membrane, moves inside the cell and then blocks ACh release. As part of our long-term on-going studies on structure, structure- function relationship and mode of action of the NT we plan for the next five years, the following: Establish complete amino acid sequences of Nt types A, B and E from proteolytic fragments of the separable L and H subunits of the 150 kDa dichain NT. Determine the conformation of the NT based on experimental (secondary and tertiary structures) and predictive methods. For 3-D structure based on x-ray diffraction, crystallization of sigma100% pure NT, now available, will be attempted. Interaction of various segments of the three NTs with lipid membranes will be studied with radiolabeled lipid analogs and by searching for lipid binding amphiphilic alpha-helices throughout the entire length of the NT. Role of the two subunits in toxicity will be tested by i) producing reconjugated dichain NTs with l and H chains from different serotypes, and ii) enzymatically removing the Cys from the H chain that forms the -S-S- between the two subunits. The presumed intracellular catalytic activity of the NT will be studied by following the """"""""spontaneous cleavage"""""""" of the separated, pure L chain and binding of nucleotides (e.g. NAD) to the NT. The clostridial protease(s) that nicks the single chain NT to the dichain form and activates it will be isolated. Arg, His and Tyr residues, that are """"""""critical"""""""" for toxicity will be located in the primary structure of the NT. Radiolabel and biotin will be covalently placed on selected locations (at non-critical Lys residues) of type A NT. The NTs produced by butyricum and barati will be partially characterized physiochemically to compare with the recognized serotypes.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS017742-10
Application #
3397819
Study Section
Toxicology Subcommittee 2 (TOX)
Project Start
1982-07-01
Project End
1995-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
10
Fiscal Year
1992
Total Cost
Indirect Cost

  Institution

Name
University of Wisconsin Madison
Department
Type
Schools of Earth Sciences/Natur
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715

  Publications

DasGupta, Bibhuti R (2006) Botulinum neurotoxins: perspective on their existence and as polyproteins harboring viral proteases. J Gen Appl Microbiol 52:1-8
Dasgupta, Bibhuti R; Antharavally, Babu S; Tepp, William et al. (2005) Botulinum neurotoxin types A, B, and E: fragmentations by autoproteolysis and other mechanisms including by O-phenanthroline-dithiothreitol, and association of the dinucleotides NAD(+)/NADH with the heavy chain of the three neurotoxins. Protein J 24:337-68
Prabakaran, S; Tepp, W; DasGupta, B R (2001) Botulinum neurotoxin types B and E: purification, limited proteolysis by endoproteinase Glu-C and pepsin, and comparison of their identified cleaved sites relative to the three-dimensional structure of type A neurotoxin. Toxicon 39:1515-31
Flicker, P F; Robinson, J P; DasGupta, B R (1999) Is formation of visible channels in a phospholipid bilayer by botulinum neurotoxin type B sensitive to its disulfide? J Struct Biol 128:297-304
Antharavally, B; Tepp, W; DasGupta, B R (1998) Status of Cys residues in the covalent structure of botulinum neurotoxin types A, B, and E. J Protein Chem 17:187-96
Antharavally, B S; DasGupta, B R (1998) Covalent structure of botulinum neurotoxin type B;location of sulfhydryl groups and disulfide bridge and identification of C-termini of light and heavy chains. J Protein Chem 17:417-28
Lawrence, G W; Foran, P; Mohammed, N et al. (1997) Importance of two adjacent C-terminal sequences of SNAP-25 in exocytosis from intact and permeabilized chromaffin cells revealed by inhibition with botulinum neurotoxins A and E. Biochemistry 36:3061-7
Beecher, D J; DasGupta, B R (1997) Botulinum neurotoxin type A: limited proteolysis by endoproteinase Glu-C and alpha-chymotrypsin enhanced following reduction; identification of the cleaved sites and fragments. J Protein Chem 16:701-12
Antharavally, B S; DasGupta, B R (1997) Covalent structure of botulinum neurotoxin type E: location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains. J Protein Chem 16:787-99
Emanuel, P; O'Brien, T; Burans, J et al. (1996) Directing antigen specificity towards botulinum neurotoxin with combinatorial phage display libraries. J Immunol Methods 193:189-97

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