Mapping of the regions on the heavy chain of botulinum neurotoxin A (BoNT/A) recognized by antibodies of cervical dystonia patients with immunoresistance to BoNT/A
Introduction
Botulinum neurotoxins (BoNTs) act on the nervous system (Dickson and Shevky, 1923a, Dickson and Shevky, 1923b) and cause paralysis by blocking the release of acetylcholine (ACh) from nerve terminals at the neuromuscular junction. Intramuscular injection of BoNTs produces a reversible partial paralysis of the selected neuromuscular junctions. Because of this activity, the toxins are employed in the treatment of a variety of clinical conditions associated with involuntary muscle spasm and contractions as well as cosmetic and other therapeutic applications (Jankovic, 2004a, Atassi and Oshima, 1999, Silberstein, 2001, Borodic et al., 1996, Borodic et al., 2001, Becker-Wegerich et al., 2002, Binder et al., 2002, Turton et al., 2002, Gui et al., 2003). But the therapeutic benefits are of finite duration and periodic injections are required. In some patients, the treatment elicits blocking Ab (and/or T cell) responses against the toxin, which reduce or completely prevent the patient's responsiveness to further treatment (Göschel et al., 1997, Atassi and Oshima, 1999, Jankovic, 2002, Jankovic, 2004b, Atassi, 2004, Jankovic et al., 2005). Recent reports, however, have shown (Jankovic et al., 2003, Comella et al., 2004) that the immune response rate to the current BoNT/A preparation available in has been substantially reduced.
The presence of blocking Abs can be screened by an in vivo, mouse protection assay (MPA), which determines the ability of a fixed volume of the serum to protect recipient mice against a lethal dose (LD100) of BoNT/A. However, the submolecular regions on BoNT/A that are recognized by blocking Abs in immunoresistant patients are not known. Molecular understanding of the recognition features would be crucial for the development of an antigen-specific manipulation or control strategy for immunoresistance.
The binding of BoNTs A and B to cell surface receptor appears to be a function of the H chain (Das Gupta and Sugiyama, 1972, Nishiki et al., 1994, Nishiki et al., 1996a, Nishiki et al., 1996b, Kozaki et al., 1989, Simpson, 1986, Simpson, 1989, Bandyopadhyay et al., 1987, Li and Singh, 1999), and the L chain, which is a zinc endopeptidase (Fu et al., 1998), is required for intracellular activity. It is now well established that the H chain binds to the acceptor thereby allowing the L chain, or a combination of H and L chains to be internalized and cause paralysis.
In recent studies we mapped the regions on the H chain that are involved in BoNT/A binding to mouse brain synaptosomes (snps) (Maruta et al., 2004), as well as the regions that bind blocking anti-BoNT/A Abs from mouse and other species (Atassi et al., 1996, Atassi and Dolimbek, 2004, Dolimbek et al., 2005). These localizations were achieved by making a panel of 60 uniform-size synthetic overlapping peptides that encompassed the entire 848-residue H chain and determining their abilities to bind snps or labeled anti-BoNT/A Abs. In the present work, we used this panel of 60 peptides to map 28 MPA-positive sera, from CD patients who had developed resistance to further BoNT/A (BOTOX®) treatment, for the regions that are recognized by the blocking Abs.
Section snippets
Botulinum neurotoxin and peptides
The synthesis, purification and characterization of the H-chain peptides used here (Table 1) have been previously reported (Atassi and Dolimbek, 2004). Active botulinum neurotoxin (strain A Hall) and formaldehyde-inactivated BoNT/A (toxoid) were obtained from Metabiologics (Madison, WI, USA). The formaldehyde was removed from the toxoid by dialysis against 0.1 M sodium phosphate buffer/0.05 M NaCl, pH 7.4 in Spectrapor membrane with 3500-molecular weight cut-off (Spectrum Medical Industries, Los
Data analysis
Initial studies on the binding of CD Abs to the synthetic H-chain peptides showed that the amounts of label bound by certain peptides were essentially the same as the amount of radiolabel bound to unrelated proteins and peptides. These non-Ab binding H chain peptides (e.g., N2, N3, N5, N6, N7, N9, N10, N11, N12, etc, see Table 2) presented an additional convenient control that is internal for each serum. Therefore, for each serum it was possible to express the binding as a ratio of the amount
Discussion
We have employed here a comprehensive synthetic peptide strategy, previously introduced and developed in this laboratory (Kazim and Atassi, 1980, Kazim and Atassi, 1982) to map on the entire H-subunit of BoNT/A for the full recognition profiles of blocking Abs from CD patients that have developed unresponsiveness to BoNT/A treatment. It had also been determined (Bixler and Atassi, 1983, Bixler and Atassi, 1984, Kazim and Atassi, 1980, Kazim and Atassi, 1982), that a five-residue overlap is
Acknowledgments
This work was supported by a grant from Allergan and by the Welch Foundation due to the award to M. Z. Atassi of the Robert A. Welch Chair of Chemistry.
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2018, VaccineSubmolecular recognition regions of the H<inf>N</inf> domain of the heavy chain of botulinum neurotoxin type A by T cells from toxin-treated cervical dystonia patients
2016, Journal of NeuroimmunologyCitation Excerpt :Thus, lower cumulative dose group showed slightly higher overall positive responses that accompanied slightly higher average SI values. We have recently mapped the Ab recognition regions on the H chain by MPA-positive serum samples from a separate group of BoNT/A-treated CD patients (Dolimbek et al., 2007). Table 6 compares the HN regions that are recognized by BoNT/A-specific T cells from BoNT/A-treated CD samples (n = 14) and from Ab-positive CD PBL samples (n = 5), and by Abs from MPA-positive CD samples (n = 28).
Submolecular recognition of the C-terminal domain of the heavy chain of botulinum neurotoxin type A by T cells from toxin-treated cervical dystonia patients
2016, ImmunobiologyCitation Excerpt :Rather patient PBLs appear to recognize sub-common epitope regions (examples are C1, C18 and C21; recognized by 43–57% of type A-treated samples with considerably high mean Z values between 2.68 and 3.57; 4 × 10−4 < P < 4 × 10−3) (Table 2). This contrasts with the results from Ab recognition regions within HC domain of serum samples from MPA-positive patients, in which C10, C15, C20 and C31 were recognized by most patient sera (Dolimbek et al., 2007). Thus, unlike anti-BoNT/A Abs that produced nearly universally recognized regions, BoNT/A-specific T cells produced sub-common regions only.