Epitope mapping of botulinum neurotoxins light chains

Abstract

Botulinum neurotoxins (BoNTs) are listed among the most potent biothreat agents. Simultaneously, two out of seven known serotypes of these toxins are used in medicine and cosmetics. This situation calls for development of detailed epitope maps of these toxins. Such maps will help to develop new ways for decreasing damage caused by these toxins if they were to be used as weapons while retaining the therapeutic effect of these toxins used as medicine. Here, we used a library of random fragments of DNA encoding the catalytic domain of botulinum neurotoxin serotype A to identify short epitope-forming sequences. We demonstrated that knowledge of such sequences in a BoNT of one serotype can be used for identification of epitope-forming sequences in other serotypes of BoNTs. We also demonstrated a serodiagnostic value of identified sequences and their ability to retain epitope-specific structures and trigger production of corresponding antibodies, even when they are transferred into a background of a completely alien carrier protein.

Figure 1. Relative location of epitope-forming peptides on the primary structure of light chain of botulinum neurotoxin serotype A

Identified sequences of peptides are highlighted and their names are marked by the bold text. Sequence of light chain of botulinum neurotoxin serotype A is in plain text.

Figure 2. Purification and antigenic properties of RAP-containing hybrid proteins

After purification on Ni-Sepharose, HisbioRAP derivatives were subjected to SDS PAGE and were visualized by staining with Coomassie (A), or were transferred onto a nitrocellulose membrane and treated with either rabbit anti-BoNT/A serum (B) or fraction of rabbit anti-BoNT/A serum, affinity purified on resin containing HisbioRAP-ep4s (C), and followed by treatment with anti-rabbit-horse radish peroxidase conjugate and diamine benzidine. Line 1 contains HisbioRAP-Aep2, 2 - HisbioRAP-Aep1-1, 3 - HisbioRAP, 4 – BoNT/AB-L (a derivative of BoNT/A-L whose C-terminal ep4-containing region is substituted with the corresponding region of BoNT/B-L), 5 - pre-stained molecular weight markers from Invitrogen, Inc., 6 – HisbioRAP-Aep4s, 7 - HisbioRAP-Aep1s, and 8 - HisbioRAP-random peptide.

Figure 3. Relative location of BoNTs sequences used for construction of RAP derivatives on the alignment of sequences of BoNT/A subtypes and BoNT serotypes

Sequences of BoNTs incorporated into RAP derivatives are marked by grey shadow and designated ep1s, ep4s and Zn. Yellow, green and blue colors are used to mark homologous residues, and the dashed lines represent not shown sequences located between two depicted blocks.

Figure 4. Recognition of RAP derivatives containing epitope-forming peptides by antibodies raised to different serotypes of BoNTs

After purification on Ni-Sepharose, HisbioRAP derivatives were subjected to SDS PAGE and were visualized either by staining with Coomassie (A), or were transferred onto a nitrocellulose membrane and treated with rabbit anti-BoNT/F (B), anti-BoNT/B (C), or anti-BoNT/A (D), followed by treatment with anti-rabbit-horse radish peroxidase conjugate and diamine benzidine. Line 1 contains HisbioRAP-A-Zn, 2 - HisbioRAP-FAep1s, 3 - HisbioRAP-BAep1s, 4 - HisbioRAP-Fep1s, 5 - HisbioRAP-Bep1s, 6 - HisbioRAP-Aep1s, 7 –BoNT/AB-L (a BoNT/A-L derivative whose C-terminal region is substituted with the corresponding region of BoNT/B-L), 8 - pre-stained molecular weight markers from Invitrogen, Inc.

Fig. 5. Specificities of antibodies raised to the hybrid protein carrying three different epitope-forming peptides

Purified proteins were subjected to SDS PAGE and were visualized either by staining with Coomassie (A), or were transferred onto a nitrocellulose membrane and treated with rabbit anti-BoNT/A (B), anti-BoNT/B (C), or sera of mice immunized with α–Tox/FBAep1s (D), followed by the treatment with either donkey anti-rabbit-horse radish peroxidase conjugate (B and C) or goat anti-mice-horse radish peroxidase conjugate (D). Antibodies trapped on the filter were visualized with Metal Enhanced DAB Substrate Kit (Pierce, Inc). Line 1 contains light chain of tetanus toxin, 2 – BoNT/B-L, 3 – BoNT/A-L, 4 – gfp-BoNT/ACH, 5 –HisbioRAP-A-Zn, 6 - HisbioRAP-FBAep1s and 7 - pre-stained molecular weight markers from Fermentas