Molecular immune recognition of botulinum neurotoxin B. The light chain regions that bind human blocking antibodies from toxin-treated cervical dystonia patients. Antigenic structure of the entire BoNT/B molecule

Abstract

We recently mapped the regions on the heavy (H) chain of botulinum neurotoxin, type B (BoNT/B) recognized by blocking antibodies (Abs) from cervical dystonia (CD) patients who develop immunoresistance during toxin treatment. Since blocking could also be effected by Abs directed against regions on the light (L) chain, we have mapped here the L chain, using the same 30 CD antisera. We synthesized, purified and characterized 32 19-residue L chain peptides that overlapped successively by 5 residues (peptide L32 overlapped with peptide N1 of the H chain by 12 residues). In a given patient, Abs against the L chain seemed less intense than those against H chain. Most sera recognized a limited set of L chain peptides. The levels of Abs against a given region varied with the patient, consistent with immune responses to each epitope being under separate MHC control. The peptides most frequently recognized were: L13, by 30 of 30 antisera (100%); L22, by 23 of 30 (76.67%); L19, by 15 of 30 (50.00%); L26, by 11 of 30 (36.70%); and L14, by 12 of 30 (40.00%). The activity of L14 probably derives from its overlap with L13. The levels of Ab binding decreased in the following order: L13 (residues 169–187), L22 (295–313), L19 (253–271), and L26 (351–369). Peptides L12 (155–173), L18 (239–257), L15 (197–215), L1 (1–19) and L23 (309–327) exhibited very low Ab binding. The remaining peptides had little or no Ab-binding activity. The antigenic regions are analyzed in terms of their three-dimensional locations and the enzyme active site. With the previous localization of the antigenic regions on the BoNT/B H chain, the human Ab recognition of the entire BoNT/B molecule is presented and compared to the recognition of BoNT/A by human blocking Abs.

Introduction

Botulinum neurotoxins (BoNTs) are protein toxins produced by Clostridium botulinum in seven serotypes (A–G) with many subtypes for each serotype. They are the most toxic substances known. The toxic effect commences by binding of the toxin to a cell-surface receptor followed by endocytosis of toxin–receptor complex. Inside the cell the toxin blocks neurotransmitter release at the presynaptic neuromuscular junction. Binding of BoNTs A and B to cell surface receptors is accomplished by the heavy (H) chain (Nishiki et al., 1996a, Nishiki et al., 1996b, Kozaki et al., 1989, Simpson, 1986, Bandyopadhyay et al., 1987, Li and Singh, 1999, Maruta et al., 2004, Jin et al., 2006, Chai et al., 2006, Dong et al., 2006, Mahrhold et al., 2006, Dolimbek et al., 2011). This allows the internalized L chain, which is a zinc endopeptidase (Schiavo et al., 1992, Fu et al., 1998), to cause paralysis.

The ability of BoNTs to locally produce a reversible decline of motor neuron activity at the affected neuromuscular junctions, provided a way for their use (mostly BoNTs A and B) in treatment of a variety of clinical conditions related to involuntary muscle spasm and contractions, and in cosmetic and other therapeutic applications (Atassi and Oshima, 1999, Binder et al., 2002, Turton et al., 2002, Gui et al., 2003, Diamond and Jankovic, 2006). Cervical dystonia (CD) is one among the conditions that are amenable to BoNT treatment. CD is associated with neck-muscle spasms that produce pain and involuntary contractions causing abnormal neck movements and posture (Jankovic 2004). The durations of therapeutic benefits, however, limited and repeat injections are needed, usually every 3–6 months. Recurring injections could educe in some patients (less with BoNT/A than BoNT/B) blocking Abs against the treating toxin, which diminish the benefit or create a complete resistance to treatment (Göschel et al., 1997, Atassi and Oshima, 1999, Jankovic, 2004, Jankovic, 2006, Jankovic et al., 2006, Atassi, 2004, Atassi, 2006). In a study of 100 patients with CD, one-third of the patients who were negative for BoNT/B Abs at baseline became positive for BoNT/B Abs during a 42-month follow up which included about 5 treatment visits per patient (Jankovic et al. 2006). Thus the high antigenicity of BoNT/B limits its long-term efficacy.

Blocking Abs are directed predominantly against regions on the H chain (Kozaki et al., 1989, Byrne and Smith, 2000, Atassi et al., 2005, Dolimbek et al., 2005, Dolimbek et al., 2007, Maruta et al., 2006). In recent publications we reported the regions on the H chain of BoNTs A and B that bind blocking Abs of CD patients treated with the correlate toxin (Dolimbek et al., 2007, Atassi et al., 2008). However, it has also been known that some blocking Abs are directed against the L chain (Adekar et al., 2008, Takahashi et al., 2009, Dolimbek et al., 2011). Therefore the purpose of the present work was to determine the binding specificity to L chain regions of blocking anti-BoNT/B Abs obtained from non-responsive CD patients. Knowledge of the targets of blocking anti-BoNT Abs affords a means for epitope-selective management of the Ab responses and for the design of peptide-based synthetic vaccines.