title: Novel ganglioside-mediated entry of botulinum neurotoxin serotype D into neurons.
abstract: Botulinum Neurotoxins (BoNTs) are organized into seven serotypes, A-G. Although several BoNT serotypes enter neurons through synaptic vesicle cycling utilizing dual receptors (a ganglioside and a synaptic vesicle-associated protein), the entry pathway of BoNT/D is less well understood. Although BoNT/D entry is ganglioside-dependent, alignment and structural studies show that BoNT/D lacks key residues within a conserved ganglioside binding pocket that are present in BoNT serotypes A, B, E, F, and G, which indicate that BoNT/D-ganglioside interactions may be unique. In this study BoNT/D is shown to have a unique association with ganglioside relative to the other BoNT serotypes, utilizing a ganglioside binding loop (GBL, residues Tyr-1235-Ala-1245) within the receptor binding domain of BoNT/D (HCR/D) via b-series gangliosides, including GT1b, GD1b, and GD2. HCR/D bound gangliosides and entered neurons dependent upon the aromatic ring of Phe-1240 within the GBL. This is the first BoNT-ganglioside interaction that is mediated by a phenylalanine. In contrast, Trp-1238, located near the N terminus of the ganglioside binding loop, was mostly solvent-inaccessible and appeared to contribute to maintaining the loop structure. BoNT/D entry and intoxication were enhanced by membrane depolarization via synaptic vesicle cycling, where HCR/D colocalized with synaptophysin, a synaptic vesicle marker, but immunoprecipitation experiments did not detect direct association with synaptic vesicle protein 2. Thus, BoNT/D utilizes unique associations with gangliosides and synaptic vesicles to enter neurons, which may facilitate new neurotoxin therapies.
authors.affiliation: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Abby R
authors.initials: AR
authors.lastname: Kroken
authors.forename: Andrew P-A
authors.initials: AP
authors.lastname: Karalewitz
authors.forename: Zhuji
authors.initials: Z
authors.lastname: Fu
authors.forename: Jung-Ja P
authors.initials: JJ
authors.lastname: Kim
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Barbieri
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D005732:Gangliosides / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D017433:Protein Structure, Secondary; D051381:Rats
chemical_list: D005732:Gangliosides; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; C489618:Sv2a protein, rat; C503969:Synpo protein, rat; C096325:botulinum toxin type D; D001905:Botulinum Toxins