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Volume 272, Number 48, Issue of November 28, 1997 pp. 30380-30386
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, May 5, 1997, and in revised form, September 22, 1997)

Robert E. Shapiro , Chelsea D. Specht ^¶ , Brian E. Collins ^¶ , Amina S. Woods ^¶ , Robert J. Cotter ^¶ and Ronald L. Schnaar ^¶**

From the Departments of  Neurology, ^¶ Pharmacology and Molecular Sciences, and ^** Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Tetanus toxin entry into vertebrate motorneurons may involve binding of neuronal surface gangliosides containing the "1b" substructure (a NeuAc2,8NeuAc 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 G_D1b (¹²⁵I-azido-G_D1b) 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 (G_T1b), but not by a non-1b series ganglioside (G_M3). When tetanus toxin C fragment was proteolyzed with clostripain, whether before or after reaction with ¹²⁵I-azido-G_D1b, a radiolabeled band was observed by SDS-polyacrylamide gel electrophoresis autoradiography, which was selectively inhibited by G_T1b. 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.

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