Bibhuti R. DasGupta

Crystal structure of botulinum neurotoxin type A and implications for toxicity.

Botulinum neurotoxin type A (BoNT/A) is the potent disease agent in botulism, a potential biological weapon and an effective therapeutic drug for involuntary muscle disorders. The crystal structure of the entire 1,285 amino acid di-chain neurotoxin was determined at 3.3 Å resolution. The structure reveals that the translocation domain contains a central pair of αhelices 105 Å long and a ~50 residue loop or belt that wraps around the catalytic domain. This belt partially occludes a large channel leading to a buried, negative active site — a feature that calls for radically different inhibitor design strategies from those currently used. The fold of the translocation domain suggests a mechanism of pore formation different from other toxins. Lastly, the toxin appears as a hybrid of varied structural motifs and suggests a modular assembly of functional subunits to yield pathogenesis.

Botulinum neurotoxins serotypes A and E cleave SNAP‐25 at distinct COOH‐terminal peptide bonds

SNAP‐25, a membrane‐associated protein of the nerve terminal, is specifically cleaved by botulinum neurotoxins serotypes A and E, which cause human and animal botulism by blocking neurotransmitter release at the neuromuscular junction. Here we show that these two metallo‐endopeptidase toxins cleave SNAP‐25 at two distinct carboxyl‐terminal sites. Serotype A catalyses the hydrolysis of the Gln197‐Arg198 peptide bond, while serotype E cleaves the Arg180‐Ile181 peptide linkage. These results indicate that the carboxyl‐terminal region of SNAP‐25 plays a crucial role in the multi‐protein complex that mediates vesicle docking and fusion at the nerve terminal.

N-ETHYLMALEIMIDE-SENSITIVE FACTOR ACTS AT A PREFUSION ATP-DEPENDENT STEP IN CA2+-ACTIVATED EXOCYTOSIS

An ATP-dependent activity of NSF (N-ethylmaleimide-sensitive factor) that rearranges soluble NSF attachment protein (SNAP) receptor (SNARE) protein complexes was proposed to be the driving force for membrane fusion. The Ca2+-activated fusion of secretory vesicles with the plasma membrane in permeable PC12 cells requires ATP; however, the ATP requirement is for a priming step that precedes the Ca2+-triggered fusion reaction. While phosphoinositide phosphorylation is a key reaction required for priming, additional ATP-dependent reactions are also necessary. Here we report that the NSF-catalyzed rearrangement of SNARE protein complexes occurs during ATP-dependent priming. NSF with α-SNAP (soluble NSF attachment protein) were required for ATP-dependent priming but not Ca2+-triggered fusion, indicating that NSF acts at an ATP-dependent prefusion step rather than at fusion itself. NSF-catalyzed activation of SNARE proteins may reorganize membranes to generate a vesicle-plasma membrane prefusion intermediate that is poised for conversion to full fusion by Ca2+-dependent mechanisms.

SNAP-25 Is Required for a Late Postdocking Step in Ca2+-dependent Exocytosis

The Ca2+-activated fusion of large dense core vesicles (LDCVs) with the plasma membrane is reconstituted in mechanically permeabilized PC12 cells by provision of millimolar MgATP and cytosolic proteins. Ca2+-activated LDCV exocytosis was inhibited completely by the type E but not the type A botulinum neurotoxin (BoNT) even though both BoNTs were equally effective in proteolytically cleaving the synaptosome-associated protein of 25 kDa (SNAP-25). The greater inhibition of exocytosis by BoNT E correlated with a greater destabilization of detergent-extracted complexes consisting of SNAP-25, synaptobrevin, and syntaxin. LDCVs in permeable PC12 cells can be poised at a late postdocking, prefusion state by MgATP-dependent priming processes catalyzed by N-ethylmaleimide sensitive factor and priming in exocytosis proteins. BoNT E completely blocked Ca2+-activated LDCV exocytosis in ATP-primed cells, whereas BoNT A was only slightly inhibitory, implying that the C-terminal region of SNAP-25 (Ile181-Gln197) between the cleavage sites for BoNT E and BoNT A is essential for late postdocking steps. A required role for SNAP-25 at this stage was also indicated by inhibition of Ca2+-activated LDCV fusion in ATP-primed cells by a C-terminal peptide antibody. We conclude that plasma membrane SNAP-25, particularly residues 181-197, is required for Ca2+-regulated membrane fusion at a step beyond LDCV docking and ATP utilization.

A common subunit structure in Clostridium botulinum type A, B and E toxins.

Abstract Tryptic activation of Clostridium botulinum type E progenitor toxin of 147,000 mol. wt. involves cleavage of the molecule into at least two polypeptides that are separable when the disulfide bond(s) linking them is reduced. The 50,000 and 102,000 mol. wt. of these chains compare with the 53,000 and 97,000 values of the disulfide-linked polypeptides of type A toxin of mol. wt. 145,000. Type E toxin resulting from trypsinization of its progenitor and naturally activated type A and B toxins have similar subunit structures.

Purification and amino acid composition of type A botulinum neurotoxin

A method to purify type A botulinum neurotoxin from a 64 liter bacterial culture is reported. The procedure includes cation exchange chromatography at pH 7.0. The final product, essentially homogeneous (according to polyacrylamide gel-sodium dodecylsulfate electrophoresis), is a mixture of two forms of the neurotoxin (mol. wt 145,000); the dichain or nicked form (over 95%) and its precursor the single chain or unnicked form. Two batches of the neurotoxin purified by the method described here and one batch purified according to the method of Sugii and Sakaguchi were similar in purity and amino acid composition. The best estimate of the number of amino acid residues per neurotoxin molecule (mol. wt 145,000) is: Asp200Thr75Ser79Glu114Pro44Gly64Ala53Val70CyS10Met22Ile111Leu104Tyr71 Phe68Lys100His14Arg43Trp17.

The N-terminal half of the heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayers

The heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayer membranes. Channel activity is confined to the N‐terminal half of this chain; the C‐terminal half is inactive. Channel activity is stimulated by low pH (4.5–5.5) on the cis side (the side to which protein is added), neutral pH on the opposite (trans) side, and cis positive voltages. These findings are strikingly similar to those previously reported for analogous fragments of diphtheria and tetanus toxins.

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

Botulinum neurotoxin (NT) serotype B, produced by Clostridium botulinum (proteolytic strain), is a ∼150-kDa single-chain polypeptide of 1291 amino acids, of which 10 are Cys residues [Whelan et al. (1992), Appl. Environ. Microbiol.58, 2345–2354] The posttranslational modifications of the gene product were found to consist of excision of only the initiating Met residue, limited proteolysis (nicking) of the 1290-residue-long protein between Lys 440 and Ala 441, and formation of at least one disulflde bridge. The dichain (nicked) protein, in a mixture with the precursor single-chain (unnicked) molecules, was found to have a ∼50-kDa light chain (Pro 1 through Lys 440) and a ∼100-kDa heavy chain (Ala 441 through Glu 1290). The limited in vivo nicking of the single-chain NT to the dichain form, by protease endogenous to the bacteria, and the nonfacile in vitro cleavage by trypsin of the Lys 440–Ala 441 bond appear to be due to the adjacent Ala 441–Pro 442 imide bonds probable cis configuration in a mixed population of molecules with cis and trans configurations. The two chains were found connected by an interchain disulfide formed by Cys 436 and Cys 445. Six other Cys residues, at positions 70, 195, 308, 777, 954, and 1277, were found in sulfhydryl form. In addition, a Cys at position 1220 or 1257 appeared to be in sulfhydryl form, hence our experimental results could not unambiguously identify presence of an intrachain disulfide bridge near the C-terminus of the NT. A total of 384 amino acid residues, including the 6 Cys residues at positions 70, 195, 308, 436, 445, and 1277, were identified by direct protein-chemical analysis; thus 29.7% of the proteins entire amino acid sequence predicted from the nucleotide sequence was confirmed. The 6 amino acids, residues 945–950, did not match with the sequence predicted in 1992, but did match with a later report of 1995. The above determinations were made by a combination of chemical (CNBr and acidic cleavage at Asp–Pro) and enzymatic (trypsin, clostripain, and pepsin) cleavages of the NT, and NT carboxymethylated with iodoacetamide (with or without 14C label), separation and isolation of the fragments by SDS–PAGE (followed by electroblotting onto PVDF membrane), and/or reversed-phase HPLC, and analyses of the fragments for the N-terminal amino acid sequences by Edman degradation and amino acid compositions.

Botulinum and Tetanus Neurotoxins

Dennis L. Leathennan and John L. Middlebrook Department of Immunology and Molecular Biology Toxinology Division United States Anny Medical Research Institute of Infectious Diseases Frederick MD 21702-5011 The phosphorylation of presynaptic proteins was examined in the presence of chlorpromazine using synaptic lysates derived from rat brain synaptosomes that had been preincubated with or without botulinum neurotoxin A. Chlorpromazine increased the phosphorylated state of a number of phosphoproteins with apparent molecular weights (MrS) between 40 and 60 kilodaltons. The drugs most prominent effect was a 2.5-3 .5-fold increase in the incoIporation of phosphate into a peptide of Mr-43 kilodaltons (pp43), as detennined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After preincubation with toxin, the chlorpromazinepromoted increase in the phosphorylated state of pp43 was inhibited in a concentrationand temperature-dependent manner.

Botulinum Neurotoxin Light Chain Inhibits Norepinephrine Secretion in PC12 Cells at an Intracellular Membranous or Cytoskeletal Site

Botulinum neurotoxin (NT) is a potent inhibitor of neurotransmitter secretion, but its intracellular mechanism and site of action are unknown. In this study, the intracellular action of NT was investigated by rendering the secretory apparatus of PC12 cells accessible to macromolecules by a recently described “cell cracking” procedure. Soluble cytoplasmic factors were depleted from permeabilized cells by washing to generate cell “ghosts” which retained cellular structural components and intracellular organelles (including secretory granules). The PC12 cell ghosts exhibited Ca2+‐activated [3H] nuorepinephrine release which was enhanced by cytosolic proteins and MgATP. PC12 cell ghosts provide the opportunity to distinguish the intracellular action of NT on soluble cytoplasmic components versus structural cellular components. The 150‐kDa NT and the 50‐kDa light chain of serotypes E and B, and to a lesser extent type A, inhibited Ca2+‐activated [3H] norepinephrine release in PC12 ghosts, but not in intact PC12 cells. The 100‐kDa heavy chain had no effect. This indicates that NT acts at an intracellular site in these cells permeabilized by “cell cracking.” The inhibition of secretion by NT was rapid and irreversible under the incubation conditions used. NT inhibition of [3H]‐ norepinephrine release from PC12 ghosts occurred in the absence of cytosolic proteins and MgATP and was not reversed by the addition of cytosolic proteins and MgATP, indicating that NT acts at an intracellular membranous or cytoskeletal site.