Abstract
We have purified and characterized recombinant botulinum neurotoxin serotype FA (BoNT/FA). This protein has also been named as a new serotype (serotype H), but the classification has been controversial. A lack of well-characterized, highly pure material has been a roadblock to study. Here we report purification and characterization of enzymatically active, and of inactive nontoxic, recombinant forms of BoNT/FA as tractable alternatives to purifying this neurotoxin from native Clostridium botulinum. BoNT/FA cleaves the same intracellular target proteins as BoNT/F1 and other F serotype BoNTs; the intracellular targets are vesicle associated membrane proteins (VAMP) 1, 2 and 3. BoNT/FA cleaves the same site in VAMP-2 as BoNT/F5, which is different from the cleavage site of other F serotype BoNTs. BoNT/FA has slower enzyme kinetics than BoNT/F1 in a cell-free protease assay and is less potent at inhibiting ex vivo nerve-stimulated skeletal muscle contraction. In contrast, BoNT/FA is more potent at inhibiting neurotransmitter release from cultured neurons.
Highlights
Botulinum neurotoxins (BoNTs) are highly potent neurotoxic proteins produced by various species of Clostridia bacteria [1]
Classification by sequence alignment correlates well with experimentally determined serotype assignments [4] and highlights a number of mosaic neurotoxins, which contain domains that align into different phylogenetic groups from each other; examples include BoNT/CD [5] and BoNT/DC [6]
The botulinum neurotoxin serotype FA (BoNT/FA) gene sequence was codon optimized for expression in E. coli and used to construct three expression plasmids encoding: (1) rBoNT/FA(0)-his, which is recombinant BoNT/FA rendered endopeptidase inactive by two point mutations in the catalytic site (E227Q and H230Y) and carrying a C-terminal ten-histidine affinity tag (prefix r indicates recombinant, (0) indicates enzymatically inactive, suffix-his indicates ten-histidine affinity tag); (2) mrBoNT/FA(0)-his, which is endopeptidase inactive recombinant BoNT/FA in which the naturally occurring activation loop is replaced by the activation loop from BoNT/F1 (K430 to L444 in UniParc sequence UPI00000B66D1); (3) mrBoNT/FA-his, which is endopeptidase active recombinant BoNT/FA containing the BoNT/F1 activation loop (Figure 1A and Supplemental Figures S1 and S2)
Summary
Botulinum neurotoxins (BoNTs) are highly potent neurotoxic proteins produced by various species of Clostridia bacteria [1]. BoNTs are classified into seven different families, called serotypes, based on their sensitivity to neutralization by various reference antisera. The serotype families are: BoNT/A, B, C1, D, E, F, and G [1,2,3]. BoNTs are classified into these serotype groups based on sequence alignment, which clusters them into phylogenetic groups, rather than by antibody neutralization experiments. Classification by sequence alignment correlates well with experimentally determined serotype assignments [4] and highlights a number of mosaic neurotoxins, which contain domains that align into different phylogenetic groups from each other; examples include BoNT/CD [5] and BoNT/DC [6].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
