Identification of a Unique Ganglioside Binding Loop within Botulinum Neurotoxins C and D-SA,

Abstract

The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G) based on a lack of cross anti-sera neutralization. BoNTs utilize gangliosides as components of the host receptors for binding and entry into neurons. Members of BoNT/C and BoNT/D serotypes include mosaic toxins that are organized in D/C and C/D toxins. One D/C mosaic toxin, BoNT/D-South Africa (BoNT/D-SA), was not fully neutralized by immunization with BoNT serotype /C or /D, which stimulated this study. Here the crystal structures of the receptor binding domains of BoNT/C, BoNT/D, and BoNT/D-SA are presented. Biochemical and cell binding studies show that BoNT/C and BoNT/D-SA possess unique mechanisms for ganglioside binding. These studies provide new information on how the BoNTs can enter host cells as well as a basis for understanding the immunological diversity of these neurotoxins.

Botulinum Neurotoxin Serotype C Associates with Dual Ganglioside Receptors to Facilitate Cell Entry

“…The hydroxyl group of Ser-1275 (OG) Table 2). The bound sialic acid, which overlapped physically with the GBP of other BoNT serotypes, was defined as ganglioside binding pocket 2 (GBP2) to reflect the similar location of but different contacts between the ganglioside and HCR than those observed for other BoNT serotypes (3).…”

“…A phenomenon observed within the BoNT/C and D family is the presence of naturally arising chimeric and mosaic toxins in addition to the prototypical serotypes (2). One mosaic toxin, BoNT/D-C is a combination of BoNT/C and BoNT/D with functional and antigenic properties that are similar but also unique relative to the parental neurotoxins (3)(4)(5)(6). BoNTs are the most toxic proteins for humans and are category A select agents.…”

“…Crystal structures of HCR/C, HCR/D, and a D-C mosaic HCR (HCR/D-C South Africa) show that although the overall architecture of the GBP is similar to other BoNT serotypes, three of the four conserved residues of the GBP (His, Ser, and Trp) are absent (3). Further analysis has identified a ␤-hairpin loop that contained a Trp residue with HCR/C, HCR/D, and HCR/D-C South Africa.…”

“…Further analysis has identified a ␤-hairpin loop that contained a Trp residue with HCR/C, HCR/D, and HCR/D-C South Africa. This ␤-hairpin loop, which aligns near the SRS and is required for ganglioside and neuron binding, is termed the ganglioside binding loop (GBL) (3,30). Additional studies have expanded our understanding of how BoNT/C and BoNT/D bind cells.…”

Enhancing the Protective Immune Response against Botulism

“…Since W is conserved within the GBPs of BoNT serotypes A, B, E, F, G and tetanus toxin (17,20,(44)(45)(46)(47), we propose that this mutation can be introduced into each HCR serotype to generate a potentially less reactive but more potent vaccine. In addition, since BoNT/C and BoNT/D possess comparable aromatic residues within another ganglioside binding loop within the HCR (48,49), we propose that the respective W and F may be modified to A to reduce potential reactivity and enhance vaccine potency. The inadvertent hazard of retaining ganglioside binding was reported during the development of the cholera toxin B subunit in adjuvant development (50).…”