Tandem fluorescent proteins as enhanced FRET-based substrates for botulinum neurotoxin activity

doi:10.1016/j.toxicon.2008.12.016

Toxicon

Volume 53, Issue 4, 15 March 2009, Pages 392-399

https://doi.org/10.1016/j.toxicon.2008.12.016 Get rights and content

Abstract

The light chain of botulinum neurotoxin A (BoNT/A-LC) is a zinc-metalloprotease that requires two extended exosites for optimal substrate binding and recognition of its intracellular target SNAP25. CFP and YFP connected through SNAP25 peptide (141–206) containing both exosites (CsY) has been used in a FRET-based assay for BoNT/A. To further improve the FRET efficiency in this BoNT/A substrate for in vitro high-throughput assays, we explored the feasibility of enhancing the capture of CFP emission by doubling the number of YFP acceptors. In comparison to CsY, the tandem fluorescence substrates CsYY and YsCsY enhanced the ratiometric fluorescence signal between YFP and CFP. YsCsY, containing two substrate sites, offered the greatest fluorometric change upon toxin-catalyzed cleavage. In addition to known approaches for enhancing fluorescence yield through various mutations, this alternative tandem substrate approach can boost the FRET signal and is particularly useful for substrates requiring extensive exosite recognition for specificity.

Introduction

Clostridial botulinum neurotoxins (BoNT), serotypes A to G, which target peripheral cholinergic neurons, are considered the most potent protein toxins for humans. Death occurs by respiratory failure caused by neuromuscular paralysis. The intravenous lethal dose of BoNT serotype A (BoNT/A), which selectively cleaves synaptosome-associated protein of 25 kDa (SNAP25) and prevents neurotransmitter release, is estimated to be 1–10 ng/kg (Arnon et al., 2001). BoNT/A is considered by the Centers for Disease Control and Prevention to be a “category A-select agent” biosecurity risk due to its potential use as a bioweapon (Arnon et al., 2001). Yet, it is also the active ingredient of BOTOX^®, which is gaining extensive use as a valuable therapeutic agent for various neuronal disorders (Montecucco and Molgo, 2005) and as a cosmetic for anti-wrinkle applications (Carruthers and Carruthers, 2001).

There is no effective antidote available to counter botulism arising from deliberate or accidental ingestion of contaminated food or misuse of BoNT-containing pharmaceuticals. Currently, the only available treatment for botulism is a combination of antitoxin immunoglobulin therapy and long-term respiratory care (Arnon et al., 2001), which in the event of mass exposure to BoNT would overwhelm current medical infrastructure. Thus, there is a critical need for the development of effective post-exposure therapies to combat botulism and speed recovery as well as for rapid and specific detection. The in vitro and in vivo disconnect found in recent high-throughput screening (HTS) of small molecule libraries for BoNT/A inhibitors (Eubanks et al., 2007) brought to light the urgent need for a quick and reliable in vitro assay for identifying physiologically relevant inhibitors.

The catalytic light chains of BoNTs (BoNT-LC) are zinc-dependent proteases that recognize extended regions of their substrates for cleavage. Recognition between BoNT/A and SNAP25 involves two extended exosites for optimal substrate binding and recognition (Breidenbach and Brunger, 2004). The minimal size of SNAP25 known to retain full activity as a BoNT/A substrate is the C-terminal 66-mer peptide (residues 141–206) with both exosites (Washbourne et al., 1997). Short peptides containing the cleavage site of the substrate can be converted into active site-based FRET substrates for inhibitor screening, but this approach would miss inhibitors specifically targeting the exosites, which are the unique features of BoNT/A necessary for distinguishing BoNT/A from other metalloproteases in cells. Dong et al. (2004) first reported the feasibility of using the CFP-YFP pair with full-length SNAP25 as a FRET-based substrate for BoNT/A in a cell-based assay or with the 66-mer peptide as a FRET substrate in an in vitro assay. However, the length of the 66-mer peptide does not allow for high FRET efficiency under optimal enzymatic cleavage conditions. We found that the simple CFP-SNAP25(141–206)-YFP substrate (CsY) provides only a small change in FRET signal after BoNT/A-LC treatment, and that this FRET signal is also highly dependent on the concentration of substrate and reaction conditions.

One obvious approach to improve the FRET in a substrate such as CsY is to shorten the linker peptide between the two fluorophores since the efficiency of FRET (E_FRET) is dependent on the distance (r) between the donor and the acceptor as described in the Förster equation: E_FRET = 1/[1 + (r/R_o)⁶], where R_o is the Förster distance at which 50% of FRET would be detected. When two fluorophores are attached to the opposite ends of the 66-mer peptide as in CsY, they are separated by more than twice the Förster distance of 48 Å for the CFP-YFP pair, presuming the peptide is folded into an alpha-helix such as that found in a SNARE complex (Sutton et al., 1998). Although shorter SNAP25-derived peptides have been reported to be substrates of BoNT/A (Schmidt and Bostian, 1997) and synthetic peptides with proper acceptor/donor pairs of fluorescent dyes, such as the commercially available SNAPtide™, have been used to monitor the activity of BoNT/A, we found that CFP-YFP tethered through a 17-mer peptide was resistant to cleavage by BoNT/A-LC (data not shown). Other strategies for optimization of FRET efficiency include use of fluorescent protein variants (Nguyen and Daugherty, 2005) or use of circularly permuted mutants of fluorescent proteins (Nagai et al., 2004).

Here, we present an alternative approach to enhance the efficiency of FRET signal by capturing the CFP emission through doubling of the YFP acceptor. As depicted in Fig. 1, when the two fluorescent proteins are far apart as in CsY, only a small fraction of the maximal FRET can be materialized due to the required length of the connecting SNAP25 peptide. However, if two YFPs are in tandem as in CsYY, then the two acceptor proteins would be at similar distances to the donor CFP, and the amount of FRET in CsYY could be nearly double that of CsY. Alternatively, as depicted in the model for YsCsY, two YFPs could be equidistantly connected to CFP through two SNAP25 peptides, likewise doubling the FRET signal, but requiring two cleavage events to lose the FRET. Our results show that joining two YFPs in tandem (CsYY) or connecting two YFPs to CFP through two separate SNAP peptides (YsCsY) nearly doubled the fluorometric changes as well as enhanced the ratiometric changes in the BoNT/A cleavage assay.

Section snippets

DNA plasmid construction

The plasmids pGFP(uv), pECFP, and pEYFP were obtained from Clonetech. pGST-SNAP25(141–206) was derived from pGST-SNAP25, which was a kind gift from Joseph Barbieri (Baldwin et al., 2004). The pGE vector was constructed from pTRC-His (Invitrogen) after removing a portion of lacI^q and inserting a T7 promoter and the GFP coding sequence after the His₆-Tag. pGFP-SNAP25(141–206) was constructed from pGE by inserting a PCR fragment encoding SNAP25(141–206) and a linker sequence between GFP and

Results and discussion

Under optimal reaction conditions with 10 μM Zn²⁺, the emission spectra of the two FRET substrates CsYY and YsCsY at concentrations of 1, 3, or 5 μM indeed showed much enhanced emission at 530 nm over that of the CsY substrate (Fig. 2A). The change of emission intensity (ΔRFU) at 530 nm after cleavage by BoNT/A-LC was proportional to the substrate concentrations used (Figs. 2 and 3A). Both CsYY and YsCsY gave ΔRFU that were greater than that for CsY. YsCsY showed the best ratiometric change of 1.6

Acknowledgements

This research was supported through funds (to B.A.W.) from the Great Lakes RCE (NIH/NIAID award 1-U54-AI-057153). We thank Joseph Barbieri for helpful discussions and critical reading of the manuscript.

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¹: These authors contributed equally to this work.

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Tandem fluorescent proteins as enhanced FRET-based substrates for botulinum neurotoxin activity

Abstract

Introduction

Section snippets

DNA plasmid construction

Results and discussion

Acknowledgements

Protein Expr. Purif.

J. Thromb. Haemost.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Curr. Opin. Pharmacol.

FEBS Lett.

J. Biomol. Screening

Botulinum toxin as a biological weapon: medical and public health management

J. Am. Med. Assoc.