Recombinant derivatives of botulinum neurotoxin A engineered for trafficking studies and neuronal delivery

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Abstract

Work from multiple laboratories has clarified how the structural domains of botulinum neurotoxin A (BoNT/A) disable neuronal exocytosis, but important questions remain unanswered. Because BoNT/A intoxication disables its own uptake, light chain (LC) does not accumulate in neurons at detectable levels. We have therefore designed, expressed and purified a series of BoNT/A atoxic derivatives (ad) that retain the wild type features required for native trafficking. BoNT/A1adek and BoNT/A1adtev are full length derivatives rendered atoxic through double point mutations in the LC protease (E224 > A; Y366 > A). ΔLC-peptide-BoNT/Atev and ΔLC-GFP-BoNT/Atev are derivatives wherein the catalytic portion of the LC is replaced with a short peptide or with GFP plus the peptide. In all four derivatives, we have fused the S6 peptide sequence GDSLSWLLRLLN to the N-terminus of the proteins to enable site-specific attachment of cargo using Sfp phosphopantetheinyl transferase. Cargo can be attached in a manner that provides a homogeneous derivative population rather than a polydisperse mixture of singly and multiply-labeled molecular species. All four derivatives contain an introduced cleavage site for conversion into disulfide-bonded heterodimers. These constructs were expressed in a baculovirus system and the proteins were secreted into culture medium and purified to homogeneity in yields ranging from 1 to 30 mg per liter. These derivatives provide unique tools to study toxin trafficking in vivo, and to assess how the structure of cargo linked to the heavy chain (HC) influences delivery to the neuronal cytosol. Moreover, they create the potential to engineer BoNT-based molecular vehicles that can target therapeutic agents to the neuronal cytoplasm.

Introduction

Botulinum neurotoxins (BoNTs)1 are a family of structurally similar proteins that cause peripheral neuromuscular blockade and respiratory paralysis, with an extremely low LD50 (1–50 ng/kg) [1]. There are 7 major serotypes (A–G) and multiple subtypes [2], but all have common structural features and a similar mechanism of action. BoNTs are synthesized as single chain propeptides (Mr approximately 150,000; approximately 1300 amino acids). The majority are activated by proteolytic cleavage to generate a disulfide-bonded heterodimer consisting of light (approximately 50 kDa) and heavy (approximately 100 kDa) chains (LC and HC). The BoNT/A heterodimer contains three functional domains. Toxicity is associated with metalloprotease activity confined to the LC; neuron binding activity is associated with the C-terminal half of the HC (HCC); and translocation activity responsible for delivering the LC protease to the neuronal cytosol is associated with the N-terminal half of the HC (HCN) [3], [4].

Although significant evidence supports a multi-step mechanism culminating in LC delivery into the neuronal cytosol, currently available methodologies have not permitted direct detection of LC in the neuronal cytosol. In the case of wt BoNTs, neuron intoxication disables further toxin uptake, and consequently LC does not accumulate to levels allowing direct visualization. Nonetheless, researchers from many laboratories, using indirect approaches, have described BoNT trafficking pathways, and have deduced how the different domains of BoNT polypeptides contribute to its unique targeting mechanism. The HC and LC of wt BoNTs can be separated, individually radiolabeled, reconstituted into the disulfide-bonded heterodimer, and subsequently used to study intracellular trafficking in neurons [5]. However because the LC–HC separation and reconstitution process results in loss of ∼90% of the toxin’s biological activity, it is difficult to conclude with confidence that the tracer localization corresponds to that of the biologically active fraction (∼10% of the radiolabeled preparation). Investigators attempting to reconstitute HC with recombinant atoxic LC likewise found that the reconstituted BoNT heterodimer had a severely reduced ability to transport LC into the neuronal cytosol [6]. Strategies to take advantage of BoNT trafficking for carrying cargo into neurons have also proven difficult to develop. Isolated wt HC has been chemically coupled to dextran, but the internalized HC adduct remained localized to the endosomal compartment and no fluorescent-labeled dextran was delivered to the neuronal cytosol [7]. These studies illustrate the difficulty of renaturing separated HCs and LCs and reconstituting native configuration including disulfide bonds. Moreover, they illustrate that chemical methods to label or attach cargo to BoNT are insufficiently selective, can produce a heterogeneous population of derivatives, and are generally too harsh to retain native BoNT activity. Such problems limit the utility of chemically labeled BoNTs as probes for definitively demonstrating BoNT trafficking pathways, or as carriers for efficiently delivering therapeutics to the neuronal cytosol. Because of these limitations, we have focused on developing genetic constructs and expression systems that enable production of full-length, disulfide-bonded, atoxic, recombinant BoNT derivatives, that retain the key structural features required for native toxin trafficking.

The large size, multi-domain structure, critical disulfide bonding and mechanical sensitivity [8] of the BoNT heterodimer make it challenging to express recombinant full-length BoNT proteins that retain native configuration and trafficking. Several laboratories have reported expression of recombinant, full-length BoNTs in Escherichia coli. Kiyatkin et al. reported the expression of BoNT/C in E. coli, with three inactivating point mutations (H229 > G; E230 > T; H233 > N) in the LC protease. This atoxic BoNT/C derivative was competent for transport across epithelia and was immunogenic when orally administered [9]. Rummel et al. described the expression of full-length single chain BoNT/G, /D, /B and /A in E. coli, either as the wt or with the LC protease inactivated by point mutation [10], [11], [12]. Pier et al. described expression of recombinant, full-length BoNT/A holotoxoid in the non-toxic Clostridium botulinum strain LNT01. The mutations R364 > A and Y366 > F were introduced into the LC (BoNT/ARYM), rendering the protein unable to cleave the substrate SNAP 25 in vitro. Immunization with this holotoxoid effectively protected mice against lethal BoNT/A challenge [13]. Webb et al. described expression of synthetic BoNT/A gene constructs optimized for codon bias in Pichia pastoris, with the mutations H223 > A; E224 > A; H227 > A to render the toxin inactive (ciBoNT/A HP), which also provided excellent protective immunity [14]. Although these latter two reports are encouraging, neither provides data on the utility of these novel recombinant BoNT derivatives for trafficking studies or for neuronal delivery.

The efforts to express recombinant BoNTs have succeeded in producing effective immunogens, which in some cases are competent for epithelial transcytosis, but none have reported the production of recombinant proteins with the structural features required for targeting the neuronal cytosol with the efficiency of wt toxins. In the work reported here, we describe the design, expression and purification of recombinant, full length, atoxic BoNT/A heterodimers that retain key structural elements required for native BoNT trafficking. Moreover, our constructs are designed to also contain a short peptide sequence that enables site selective attachment of cargo molecules under physiological conditions. All of the BoNT/A derivatives reported here were designed to be atoxic, in that they are either expressed with the LC inactivated by a double mutation or with the entire catalytic region of the LC removed, enabling their uptake and accumulation into neurons at levels allowing direct detection, trafficking studies and cargo delivery.

Section snippets

Modification of pLitmus28i by replacement of the existent polylinker with a custom polylinker

A vector with a custom polylinker derived from Litmus 28i (New England Biolabs, Cat # N3528S, 2823 bp) was used for subcloning the full-length BoNT/Aadek. This derivative of Litmus 28i (pLitmus28C1) was created by restriction digestion of Litmus 28i with Bgl II and Aat II followed by dephosphorylation. In all subcloning procedures described below, vectors were digested with the same set of restriction endonucleases as were the DNA fragments for subcloning, followed by vector dephosphorylation,

Results

The full-length BoNT/A ad (atoxic derivatives) DNA and proteins were generated under biosafety level 2 containment (project approved by CDC on 02.07.2006 for the registered entity C20060207-0419).

To improve the yield of recombinant proteins, the DNA sequence encoding the full-length construct was synthesized de novo, and optimized for expression in both Sf9 insect cells and E. coli as explained in Methods. Our attempts to express BoNT/Aadek in E. coli were not successful despite repeated

Discussion

BoNTs are large, multi-domain, disulfide-bonded heterodimers, with mutual stabilization of domains through hydrogen bonds and hydrophobic interactions [21], [22], [23]. It is therefore challenging to produce recombinant BoNT derivatives that retain structural features required for native BoNT trafficking. Factors affecting the success of protein expression include the design of expression constructs and the choice of expression system. When domains are expressed separately, they can be

Acknowledgments

We thank Dr. Eric Johnson for discussion. Support for these studies was provided by NIH-NIAID AI072466 to K.I., NIH-NINDS NS050276, NIH-NCRR RR017990 to T.A.N., and NIH Office of the Director DP2-OD004631 to T.J.C. wt BoNT/A was supplied by William Tepp from the laboratory of Dr. Eric Johnson (University of Wisconsin at Madison). pET29 vector carrying C-terminally His6-tagged Sfp phosphopantetheinyl transferase from B. subtilis was kindly provided by Dr. Jun Yin (University of Chicago). Authors

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