PMC4320087_01
Comparison of BoNT/A subtypes.. BoNT/A1 amino acid sequence of strain ATCC 3502 as prototype was compared to representatives of subtypes BoNT/A2 to A7 and the novel BoNT/A8. Sequence differences are indicated by vertical lines. The cartoon indicates the domain of the light chain (LC), the N-terminal part (HN) and the C-terminal part (HC) of the heavy chain (HC). The latter can be further subdivided into an N-terminal (HCN) and C-terminal (HCC) subdomain. The alignment illustrates that the majority of differences between BoNT/A8 and A1 are located in the heavy chain. Furthermore, the arginine insertion at position 888 is unique to BoNT/A8.
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 Fig. 1 shows a schematic representation of BoNT/A1 to BoNT/A8, with vertical lines representing differences in amino acids compared to BoNT/A1. This makes BoNT/A8 the second 1297 aa long BoNT/A subtype next to the BoNT/A2 of strain CDC2171 (Fig. 1 and S1 Fig.). Overall, BoNT/A8 showed 87 amino acid mutations compared to BoNT/A1, with 12 amino acid substitutions at positions being unique for BoNT/A8 (Fig. 2A and S1 Fig.).
Surface representation of BoNT/A8 and arginine insertion.. (A) Surface representation of BoNT/A (PDB code 3BTA). Differences of BoNT/A8 to BoNT/A1 are marked in yellow, unique amino acid differences in BoNT/A8 (based on the representatives of BoNT/A2 to A7 used in Fig. 1) are marked in red, and the arginine insertion in position 888 is marked in green. (B) Representative MS/MS spectra of the precursor ion m/z 923.01 ([M+2H]2+], inset) of BoNT/A8 with the sequence of 872NITNTSILSIVVDKDGR888, showing the additional arginine at position 888 leading to an additional trypsin cleavage site.
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Overall, BoNT/A8 showed 87 amino acid mutations compared to BoNT/A1, with 12 amino acid substitutions at positions being unique for BoNT/A8 (Fig. 2A and S1 Fig.). Noteworthy, the whole bont/a8 gene cluster:with the exception of the bont/a8 gene itself:was closest related to the bont/f1 gene cluster of strain Langeland also belonging to C. botulinum group I (Fig. 3, Table 2 and Table 3). MS/MS spectra of the doubly charged precursor ion m/z 923.01 revealed the amino acid sequence 872NITNTSILSIVVDKDGR888 and verified the arginine insertion at position 888 (Fig. 2B), which led to an additional trypsin cleavage site.
Bont/a8 gene cluster organization.. Schematic representation of the neurotoxin gene clusters from the prototype BoNT/A1 strain ATCC 3502, BoNT/A1(B) strain NCTC 2916, BoNT/A8 strain Chemnitz, and BoNT/F1 strain Langeland. The bont/a1 gene from ATCC 3502 is located within an ha
+
orfX
-cluster containing the non-toxic non-haemagglutinin (ntnha) gene, the three haemagglutinin genes (ha70, ha17 and ha33) and the regulatory botR gene. The bont/a1 neurotoxin gene cluster of ATCC 3502 is flanked by the flagellin (fla) gene and an insertion sequence element (IS). In contrast to the prototype bont/a1 (ATCC 3502), the bont/a1 of NCTC 2916 and the bont/a8 gene of Chemnitz are located in an ha
-
orfX
+ cluster which contains the adjacent ntnha gene and:instead of the ha genes:the three orfX1-3 genes; apart from the botR gene the cluster also contains the p47 gene of unknown function. The neurotoxin gene clusters of NCTC 2916 and Chemnitz are located within the ars operon on the chromosome and are flanked by the arsC and the lycA genes. Except for the toxin gene itself, its gene cluster organization and sequence is most similar to the bont/f1 gene cluster of strain Langeland.
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The bont/a8 cluster belonged to the ha - orfX + type (Fig. 3) which is known to encode BoNT/A1, A1(B), A2, A3, A4, and A6 subtypes as well as BoNT/E and F serotypes. Noteworthy, the whole bont/a8 gene cluster:with the exception of the bont/a8 gene itself:was closest related to the bont/f1 gene cluster of strain Langeland also belonging to C. botulinum group I (Fig. 3, Table 2 and Table 3).
Differential binding of antibodies to BoNT/A1 and BoNT/A8.. Native or recombinantly expressed single chain BoNT/A1 and single chain BoNT/A8 were coated onto microtiter plates and tested in an indirect ELISA for capturing a panel of monoclonal and polyclonal antibodies generated in-house. All antibodies shown are monoclonal antibodies except for AR47 and AC29 which are polyclonal antibodies derived from rabbit and chicken, respectively. Shown is the mean (+- SD) of three independent experiments performed in duplicates.
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As shown in Fig. 4, a number of monoclonal antibodies (mAbs) showed no difference in recognizing BoNT/A1 and A8, while others showed a reduced binding or even a complete loss of binding to BoNT/A8 (mAb HcA16/6, HcA78/6, HcA86/2, A324/10, and A401/3). BoNT/A1 and BoNT/A8 recognize different epitopes on the BoNT molecule, with mAb HcA16/6, HcA78/6 and HcA86/2 being directed against the HC-domain, mAb A401/3 recognizing the LC domain and mAb A324/10 recognizing a yet to be determined conformational epitope on the molecule. Binding to two polyclonal antibodies raised in rabbit (AR47) or chicken (AC29) was not affected (Fig. 4).
Binding characteristics of BoNT/A8 cell-binding domain HC.. (A) Binding affinity of HCA8 to its synaptic protein receptor rat SV2C in a GST pull-down assay is similar to HCA1. 12.5% SDS-PAGE and Coomassie staining of a representative pull-down experiment. (B) Densitometric quantification of bound HC-fragments from (A) (n = 4 +- SD). (C) Competition assay: Binding of 35S-HCA1 to synaptosomes at 4 C in the presence of increasing amounts of recombinant HCA1 or HCA8, respectively. Bound 35S-HCA1 was detected by SDS-PAGE and autoradiography (n = 3 +- SD; HCA1: EC50 = 24 nM (95% CI 20-29 nM), HCA8: EC50 = 56 nM, 95% CI 43-72 nM)).
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GT-matrix. GST-rSV2C 454-579 could equally well precipitate HCA1 and HCA8 from the supernatant, indicating no change in affinity towards their protein receptor (Figs. 5A and 5B). Whereas recombinant HCA1 displayed an EC50 of 24 nM (95% CI 20-29 nM), 2.5-fold more HCA8 was required to displace 35S-HCA1 (EC50 = 56 nM, 95% CI 43-72 nM), indicating that HCA8 has a lower binding affinity to gangliosides incorporated in neuronal membranes compared to HCA1 (Fig. 5C).
Comparison of the catalytic activity of BoNT/A1 and BoNT/A8.. (A) Quantitative mass spectrometric endopeptidase assay of native full-length BoNT/A1 and BoNT/A8 immunoenriched from cell culture supernatants tested for cleavage of a SNAP-25 peptide substrate. (B) Quantitative mass spectrometric endopeptidase assay of recombinant light chains of BoNT/A1 and BoNT/A8 tested for cleavage of human H6SNAP-25 protein. Both diagrams show the percentage of BoNT/A8 substrate cleavage compared to BoNT/A1 (n = 3 +- SD). (C) Time-dependent cleavage of rat SNAP-25H6 (3 muM) by LC/A1 and LC/A8 (0.5 nM). The amount of cleavage product was quantified densitometrically from SDS-PAGE stained by Coomassie (n = 3 +- SD).
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We found that BoNT/A8 showed only about 28% activity compared to that of BoNT/A1 (Fig. 6A). Consistent with the results obtained with the native full-length molecule we found a dramatic reduction in catalytic activity to about 10% when comparing the activity of LC/A8 to that of LC/A1 (Fig. 6B). Again, results clearly corroborated the fact that LC/A8 displays a lower rate of catalysis than LC/A1 (Fig. 6C).
Biological activity of BoNT/A1 and A8.. The biological activity of recombinant full-length active BoNT/A1 and BoNT/A8 was determined employing the mouse phrenic nerve hemidiaphragm (MPN) assay. To a three-point dose:response curve (n >= 3 +- SD) of each subtype a power function was fitted. To yield an identical paralytic halftime, six-fold higher concentrations of BoNT/A8 are required.
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Comparison of the three-point dose:response curves (Fig. 7) yielded an about six-fold reduced biological activity of BoNT/A8 versus BoNT/A1, caused by the reduced binding and catalytic properties of BoNT/A8.