Elsevier

Molecular Immunology

Volume 44, Issue 5, February 2007, Pages 1029-1041
Molecular Immunology

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

https://doi.org/10.1016/j.molimm.2006.03.011Get rights and content

Abstract

The purpose of this work was to map the entire recognition profile of the H chain of botulinum neurotoxin A (BoNT/A) by Abs in sera that have protective anti-BoNT/A Abs by the mouse protection assay (MPA) from cervical dystonia (CD) patients who had been treated with botulinum neurotoxin, serotype A (BOTOX®). In previous studies we found that human anti-tetanus neurotoxin (TeNT) Abs cross-react with BoNT/A and BoNT/B. In the present work we devised an assay procedure for measuring specific anti-BoNT/A Abs in human sera by absorbing out or inhibiting the anti-TeNT Abs with TeNT before analyzing the sera for the anti-BoNT/A Abs. The sera were obtained from 28 CD patients who had become unresponsive to treatment with BoNT/A and the sera were found to protect mice against a lethal dose of BoNT/A. For localization of the Ab-binding regions on the H chain we employed a set of sixty, 19-residue synthetic peptides (except for peptide C31 which was 22 residues) that encompassed the entire H chain sequence 449–1296 and overlapped consecutively by five residues. The pattern of Ab recognition varied from patient to patient, but a very limited set of peptides were recognized by most of the patients. These were, in decreasing amounts of Ab binding, peptide N25 (H chain residues 785–803), C9/C10 (967–985/981–999), C31 (1275–1296), C15 (1051–1069), C20 (1121–1139), N16 (659–677), N22 (743–761), and N4 (491–509). But not every serum recognized all these peptides. The finding that the binding profile was not the same for all the patients is consistent with previous observations that immune responses to protein antigens are under genetic control and that the response to each epitope within a protein is under separate genetic control. Except for the region within C9/C10, the other regions either coincided (N16 and C31), or overlapped (N4, N22, N25, C15 and C20), with the recently mapped synaptosomes (snps)-binding regions on the H chain. The molecular and clinical implications of these findings are discussed.

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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