Identification of a Ganglioside Recognition Domain of Tetanus Toxin Using a Novel Ganglioside Photoaffinity Ligand

Robert E Shapiro,Chelsea D Specht,Brian E Collins,Amina S Woods,Robert J Cotter,Ronald L Schnaar

doi:10.1074/jbc.272.48.30380

Robert E Shapiro, Chelsea D Specht + Show 4 more

Open Access

https://doi.org/10.1074/jbc.272.48.30380

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Abstract

Tetanus toxin entry into vertebrate motorneurons may involve binding of neuronal surface gangliosides containing the "1b" substructure (a NeuAcalpha2,8NeuAc group on an internal galactose residue). The domains of tetanus toxin involved in ganglioside binding are known to reside within the carboxyl-terminal half of the toxin's heavy chain ("C fragment"). We developed a novel photoaffinity reagent based upon the structure of the 1b ganglioside GD1b (125I-azido-GD1b) to define the ganglioside-binding domains of tetanus toxin. Using this ligand, we observed radiolabeling of tetanus toxin C fragment which could be specifically inhibited by a ganglioside of the 1b series (GT1b), but not by a non-1b series ganglioside (GM3). When tetanus toxin C fragment was proteolyzed with clostripain, whether before or after reaction with 125I-azido-GD1b, a radiolabeled band was observed by SDS-polyacrylamide gel electrophoresis autoradiography, which was selectively inhibited by GT1b. Protein sequencing of proteolyzed tetanus toxin C fragment co-migrating with that band revealed the carboxyl-terminal 34 amino acid residues of tetanus toxin. Matrix-assisted laser desorption/ionization mass spectrometry of a photoaffinity labeled synthetic polypeptide representing the 34-amino acid domain revealed modification at a single residue (His1293). We propose that this domain of tetanus toxin is sufficient for ganglioside binding.

Highlights

The major Clostridial neurotoxins, botulinum and tetanus toxins, are a family of homologous proteins with selective toxicity for vertebrate motorneurons [1]
When tetanus toxin C fragment was preincubated with ganglioside ganglioside of the 1b series (GT1b) prior to reaction with 125I-azidoGD1b, radiolabeling was inhibited (Fig. 3B)
Half-maximal inhibition of 125I-azido-GD1b labeling of tetanus toxin C fragment occurred at Ϸ1 ␮M GT1b

Summary

Introduction

The major Clostridial neurotoxins, botulinum and tetanus toxins, are a family of homologous proteins with selective toxicity for vertebrate motorneurons [1]. In a highly informative study, Halpern and Loftus [22] synthesized various peptide fragments near the carboxyl terminus of tetanus toxin and measured their binding to immobilized GT1b ganglioside and neurons at physiological pH and ionic strength Their data indicated that ganglioside binding is mediated by the carboxyl-terminal portion of the tetanus toxin C fragment, contributions of protein secondary and tertiary structure to binding (e.g. interactions of non-contiguous polypeptide sequences) may be significant. An advantage of azido-GD1b photoaffinity labeling is that ganglioside binding may be performed prior to toxin fragmentation, while the protein is in its native conformation Using this technique we identify the 34-amino acid peptide at the carboxyl terminus of tetanus toxin as sufficient for ganglioside binding, and demonstrate specific photoaffinity labeling at His1293

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