Accelerated Neuronal Cell Recovery from Botulinum Neurotoxin Intoxication by Targeted Ubiquitination
Shoemaker CB (2011) Accelerated Neuronal Cell Recovery from Botulinum Neurotoxin Intoxication by Targeted Ubiquitination. PLoS
ONE 6(5): e20352. doi:10.1371/journal.pone.0020352
Accelerated Neuronal Cell Recovery from Botulinum Neurotoxin Intoxication by Targeted Ubiquitination
Chueh-Ling Kuo 0
George A. Oyler 0
Charles B. Shoemaker 0
Nic D. Leipzig, The University of Akron, United States of America
0 1 Department of Biomedical Sciences, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, United States of America, 2 Synaptic Research LLC , Baltimore, Maryland , United States of America
Botulinum neurotoxin (BoNT), a Category A biodefense agent, delivers a protease to motor neuron cytosol that cleaves one or more soluble NSF attachment protein receptors (SNARE) proteins involved in neurotransmission to cause a flaccid paralysis. No antidotes exist to reverse symptoms of BoNT intoxication so severely affected patients require artificial respiration with prolonged intensive care. Time to recovery depends on toxin serotype because the intraneuronal persistence of the seven known BoNT serotypes varies widely from days to many months. Our therapeutic antidote strategy is to develop 'targeted F-box' (TFB) agents that target the different intraneuronal BoNT proteases for accelerated degradation by the ubiquitin proteasome system (UPS), thus promoting rapid recovery from all serotypes. These agents consist of a camelid heavy chain-only VH (VHH) domain specific for a BoNT protease fused to an F-box domain recognized by an intraneuronal E3-ligase. A fusion protein containing the 14 kDa anti-BoNT/A protease VHH, ALcB8, joined to a 15 kDa F-box domain region of TrCP (D5) was sufficient to cause increased ubiquitination and accelerate turnover of the targeted BoNT/A protease within neurons. Neuronal cells expressing this TFB, called D5-B8, were also substantially resistant to BoNT/ A intoxication and recovered from intoxication at least 2.5 fold quicker than control neurons. Fusion of D5 to a VHH specific for BoNT/B protease (BLcB10) led to accelerated turnover of the targeted protease within neurons, thus demonstrating the modular nature of these therapeutic agents and suggesting that development of similar therapeutic agents specific to all botulinum serotypes should be readily achievable.
Funding: This project was funded in part with federal funds from the NIAID, NIH, DHHS, under Award Numbers U54 AI057159 and R21 AI088489, and Contract
No. N01-AI-30050. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIAID and NIH. These funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. GO is an employee of Synaptic Research LLC and
contributed to the original concept and the data analysis.
Competing Interests: GO is an employee of Synaptic Research LLC. A patent application on some technology reported in this manuscript has been submitted
by Tufts University that includes the authors as inventors. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials, as
detailed online in the guide for authors.
Botulism is caused by exposure to Clostridium botulinum neurotoxin
(BoNT), a CDC Category A biodefense threat agent for which no
antidote exists to reverse the symptoms of paralysis after onset.
Intoxication is caused when the BoNT protease light chain (Lc)
domain is delivered to the presynaptic terminal of motor neurons by
the heavy chain (Hc) domain. In the presynaptic terminal the Lc
cleaves SNARE proteins and inactivates neurotransmission
[1,2,3,4,5,6,7,8]. Seven different BoNT serotypes have been
discovered to date (BoNT/A-G). The Lc proteases of the seven
different BoNT serotypes have distinct active sites that cleave
different sites in one or more SNARE proteins [3,4,9,10,11]. Thus,
to protect against all known forms of BoNT, conventional small
molecule drug development would need to be separately performed
for each of the seven different drug targets, and perhaps even some
of the subtypes. This challenge, together with other extreme hurdles
confronting BoNT small molecule drug development, seriously
complicates efforts to develop agents to treat botulism. New
therapeutic paradigms are urgently needed to counter the enormous
risks associated with these easy-to-obtain, easy-to-produce and
extremely dangerous bioterror agents.
It is known that persistence of the symptoms of botulism varies
dramatically following intoxication by different BoNT serotypes
. BoNT/A, the serotype with the longest persistence, has
proven the most useful for therapeutic applications but also is
considered the most dangerous as a biodefense threat. Persistence
of symptoms has been related to prolonged survival of the Lc in
the presynaptic terminal . We reported evidence that this
variation is due to the variable susceptibility of different BoNT Lcs
to ubiquitination and proteasome-mediated turnover .
Furthermore, we showed that targeted ubiquitination of BoNT
protease accelerated its turnover in neuroblastoma cells .
The biomolecules employed were large and not very specific for
the BoNT protease and thus not practical for therapeutic use.
Here we report development of biomolecules that are highly
specific for BoNT proteases, small and stable enough to be
practical for therapeutic use, and capable of accelerating BoNT
protease turnover leading to a more rapid molecular cure of
Our therapeutic strategy builds on the demonstration by Zhou
et al.  that a fusion protein of the F-box protein, b-TrCP, and
an artificial protein binding domain can target a naturally stable
protein for rapid proteasomal degradation. b-TrCP associates with
Skp1 and Cullin to form the SCF complex, a multimeric E3
ubiquitin-ligase [16,17] previously shown to be expressed in
neuronal cells . F-box proteins like b-TrCP contain two
modular domains: a protein-protein interaction domain for
binding substrates and the F-box which is required for association
into the E3-ligase complex [19,20]. Using this concept, we sought
to engineer an artificial F-box protein containing a minimal F-box
domain from b-TrCP and a small targeting domain that
specifically binds to BoNT proteases.
The antigen binding VH region of camelid heavy-chain-only
antibodies, also called VHHs, were used as the BoNT LC protease
targeting domain [21,22]. VHHs are small, stable, well-expressed
proteins that bind their target with high affinity and specificity,
have excellent solubility properties, and often are potent inhibitors
of target protein function [21,23,24,25]. We previously reported
the identification of high affinity VHHs (,10 nM KD) that
recognize the proteases from either BoNT/A or BoNT/B, and
demonstrated that these VHHs retain their binding properties
within neuronal cell cytosol . Here we show that fusions of
these VHHs to a minimal F-box domain, called targeted F-box
(TFB) agents, effectively promote turnover of BoNT/A or BoNT/
B proteases and accelerate neuronal recovery from symptoms of
BoNT intoxication. Because of the modular nature of these
antidotes, it should be straightforward to develop similar agents
targeting all seven BoNT serotypes and subtypes by substituting
the VHH with other VHHs having the appropriate specificity.
Ideally, these TFB agents would be delivered to intoxicated
neurons in botulism patients by a neuronally targeted delivery
vehicle; for example as fusions to an atoxic mutant form of BoNT.
If successful, such therapy would lead to shortened persistence of
paralysis in botulism patients, thereby reducing the danger posed
by these potential terror agents.
Fusions of TrCP F-box to a BoNT Lc-specific VHH
specifically reduce steady-state Lc expression levels
within neuroblastoma cells
We previously demonstrated that the camelid heavy-chain-only
VH (VHH), ALcB8, binds to BoNT/A Lc protease (ALc) within
neuronal cells and inhibits its protease activity . The ALcB8
VHH was expressed as a fusion protein with the F-box protein,
TrCP, to create a targeted F-box (TFB) designed to promote the
specific, SCF E3-ligase mediated polyubiquitination of ALc and
consequent proteasome-mediated degradation . Initially, TFB
function was measured indirectly through ALc activity since the
exceedingly low level of ALc within intoxicated neuronal cells
made it impractical to directly measure turnover. The B8-TrCP
TFB fusion protein or ALcB8 alone were expressed within BoNT/
A intoxicated neuroblastoma Neuro 2A (N2A) cells together with
the ALc substrate, SNAP25, expressed as an indicator protein
flanked by yellow fluorescent protein (YFP) and cyan fluorescent
protein (CFP) . Cells expressing B8-TrCP were reproducibly
found to prevent cleavage of the co-transfected indicator protein,
and more effectively than ALcB8 alone (Figure S1A). The
B8TrCP TFB was itself heavily polyubiquitinated in N2A cells and its
steady state expression level was thus very low (Figure S1B),
especially compared to ALcB8. This indicated that the ability of
B8-TrCP to reduce ALc activity in intoxicated cells was due to
accelerated turnover rather than protease inhibition.
The F-box domain within TrCP that is required for association
with Skp1 within the SCF E3-ligase complex  is only about 50
amino acids. A series of expression vectors (represented in
Figure 1A) were prepared to identify the minimum portion of
TrCP required to retain TFB function. Steady-state expression
levels of the B8-TrCP fusion protein were not much improved by
removal of the TrCP 39 untranslated region (UTR) alone (B8-D1)
or most of the TrCP WD40 repeats (B8-D2) (Figure 1B). Removal
of all TrCP WD40 repeats (B8-D3), though, resulted in much
higher steady-state expression of the TFB and also shifted the
predominant sub-cellular localization from the typical TrCP
nuclear site to the cytosol as previously observed [27,28] (Figure
S2). Deletions of additional regions of TrCP flanking the F-box
domain (B8-D4, B8-D5) also displayed improved steady-state
expression levels (Figure 1B) and cytosol localization (Figure S2).
Swapping the VHH and F-box domains (D3-B8, D5-B8,
Figure 1A) did not significantly alter expression levels or
localization. Expression of all TFBs in which the ALcB8 was
fused to the TrCP F-box region present in D5 (aa 175293)
protected N2A cells from BoNT/A cleavage of SNAP25 (Figure
S3). A further truncation of TrCP (aa 175233) produced variable
results and was not pursued.
A second TFB was produced in which the ALcB8 VHH
targeting domain was replaced with a VHH (BLcB10) having
specificity for BoNT/B Lc (BLc) . The TrCP F-box domain,
D5, was fused in frame with BLcB10 in the orientation in which
the VHH is at the carboxyl terminus (D5-B10, Figure 1A). The
D5-B10 protein was expressed in the neuroblastoma cell line,
M17, and shown to retain the ability to bind BLc in cells based on
pull-down assays (Figure S4).
The ALc-specific TFB, D5-B8, and the BLc-specific TFB,
D5B10, were co-expressed within N2A cells together with ALc and/
or BLc and tested for their influence on Lc steady-state levels.
Expression of D5-B8 in N2A cells reduced steady-state levels of
ALc compared to cells expressing D5-B10 (Figure 2A). In contrast,
levels of BLc were lower in N2A cells co-expressing D5-B10 than
those expressing D5-B8. The protein levels for ALc were
quantified by capture ELISA and shown to be reduced about
65% in N2A cells that co-expressed the ALc TFB, D5-B8, as
compared to D5-B10 (Figure 2B). N2A cells expressing the BLc
TFB, D5-B10, contained about 50% of the BLc level found in cells
expressing D5-B8 (Figure 2B). Similar results were obtained when
both ALc and BLc were expressed in the same N2A cells
expressing either D5-B8 or D5-B10 (Figure S5). SNAP25 served as
the loading control in these experiments. These results
demonstrate the modular nature of the TFBs in which the VHH domains
can be exchanged with other VHHs having a different specificity
and thereby target a different protein for accelerated degradation.
TFBs promote target-specific ubiquitination
CFP-ALc was co-expressed with the TFBs D5-B8 or D5-B10 in
the presence of the proteasome inhibitor, MG132, to permit
accumulation of polyubiquitinated proteins. The cells were also
co-transfected with an expression plasmid for HA-ubiquitin. The
CFP-ALc was purified from cell extracts by affinity to
GSTALcB8. The use of the VHH ALcB8 to purify the ALc should
eliminate (by competition) co-purification of any D5-B8 that
remained bound to the CFP-ALc in the extract and which could
lead to contaminating polyubiquitinated protein. The purified ALc
from each extract was analyzed by Western blot (Figure 3) and
shown to contain CFP-ALc. The amount of extract loaded was
normalized such that the CFP-ALc levels were nearly the same.
When an identical Western blot was analyzed for HA, it became
clear that the ALc co-expressed with D5-B8 was much more
heavily ubiquitinated than ALc co-expressed with D5-B10. These
results show that the TFB D5-B8 is promoting ubiquitination of
ALc within the transfected M17 cells.
Figure 1. Expression of Lc-targeting TFBs containing various truncations of TrCP cDNA. (A) Schematic of various expressed TFB fusion
proteins containing different regions of TrCP cDNA including the F-box domain (scale approximate). All fusion proteins contain a streptavidin binding
peptide (SBP) and a yellow fluorescent protein (YFP) domain at the amino end. Each TFB contains the BoNT/A Lc binding VHH, ALcB8, or the BoNT/B
Lc binding VHH, BLcB10. Some proteins contain one or more WD40 repeat (w). (B) Western blot of recombinant TFBs expressed in M17 cells. M17 cells
were transfected with expression plasmids for the different ALcB8 TFB protein diagrammed in A. The proteins were affinity purified with GST-ALc,
resolved by SDS-PAGE and detected by Western blot with anti-GFP antibody (Santa Cruz). Data shown is representative of three separate
experiments. The 117 kDa B8-TrCP and B8-D1 proteins were apparent on longer exposures.
TFBs accelerate protein turnover in a target-specific
M17 stable cell lines that express a transgene for either the TFB
D5-B8 or D5-B10 were created by lentivirus vector transduction.
Virtually all cells in these populations express the TFB transgene
based on YFP fluorescence. The D5-B8 and D5-B10 cell lines
were transfected with an expression plasmid for CFP-ALc and the
level of ALc expression (and p47 as a loading control) was detected
at various times post-transfection by Western blot (Figure 4A) and
quantified by scanning (Figure 4B). The apparent half life for
CFPALc when co-expressed with D5-B8 (ALc TFB line) was ,1.5 days
while in cells co-expressing D5-B10 (BLc TFB line), the half life of
CFP-ALc was ,3.7 days. The studies clearly show that D5-B8
accelerated the turnover of CFP-ALc (p,0.005). In a separate
experiment, the levels of ALc, TFB and SNAP25 were each
individually assessed by Western blot at days 3, 4 and 5 (Figure
S6). Once again, the ALc levels were reduced much more rapidly
when co-expressed with D5-B8 compared to D5-B10 while the
levels of the stably expressed TFB and endogenous SNAP25
remained nearly constant as expected. The efficacy of TFBs to
accelerate Lc turnover was dependent on proteasome function as
no differences in ALc and BLc steady-state levels were observed in
D5-B8 or D5-B10 cells treated with the proteasome inhibitor,
MG132 (Figure S7).
M17 cells that stably express the TFB D5-B8 or D5-B10 were
compared for their susceptibility to BoNT/A intoxication as
assessed by cleavage of SNAP25. The D5-B8 cells were found to
become intoxicated to a significantly lesser degree (,30%
SNAP25 cleavage) than D5-B10 cells (Figure S8) or parental
M17 cells (7080% SNAP25 cleavage) (p,0.005). As the VHH
ALcB8 component of D5-B8 is a potent inhibitor of ALc protease
, it is not possible to separate the contributions of protease
inhibition and accelerated ALc turnover in this assay.
ALc TFB (D5-B8) promotes accelerated recovery of M17
cells following BoNT/A intoxication
Finally we tested whether the levels of intact SNAP25 recover
more rapidly following BoNT/A intoxication of neuroblastoma
cells when expressing the ALc-targeting TFB, D5-B8. M17 cell
lines that constitutively express either TFB D5-B8 or D5-B10 were
intoxicated with BoNT/A. Cells were nearly confluent at the time
of intoxication to limit new cell division that might dilute the
intoxication effect. At various times post-intoxication, cells were
harvested and assessed for the proportion of intact SNAP25
(Figure S9). With time, the proportion of intact SNAP25 recovered
to some extent in all cases. In cells expressing D5-B8, intact
SNAP25 recovered to near pre-intoxication levels in two weeks
(Figure 5). Within control M17 cells or M17 cells expressing TFB
D5-B10, intact SNAP25 represented less than 50% of the total
SNAP25 after two weeks. In these experiments, the presence of
D5-B8 promoted about 2.5 fold more rapid recovery vs. both
controls and the difference was highly significant (p,0.001).
Figure 2. BoNT Lc targeted TFBs reduce the steady-state level of co-expressed Lc in transfected neuroblastoma cells. N2A cells were
co-transfected with expression vectors for CFP-ALc or CFP-BLc and for TFB D5-B8 or D5-B10 as indicated. (A) Western blots. 24 hrs post-transfection,
cell extracts were prepared and resolved by SDS-PAGE. CFP-Lc and SNAP25 expression levels were detected by Western blotting using anti-GFP or
anti-SNAP25 antibody. (B) Capture ELISA. Cell extracts prepared from transfected cells in A were quantified by capture ELISA. Background absorbance
was subtracted from the absorbance at OD450 nM. Data are presented as averages 6 standard deviation calculated from three independent samples
and compared by unpaired t test. The differences between each pairing are highly significant (p,0.001).
The therapeutic challenges of Botulinum neurotoxin poisoning
are largely due to its extreme potency and the long persistence of
the resulting flaccid paralysis. Currently there is no antidote for the
symptoms of botulism once paralysis has become established.
Development of small molecule drugs that inhibit BoNT proteases
to reverse botulism is feasible, but faces enormous challenges. For
example, at least seven BoNT serotypes exist, each having a
protease with different substrate specificity, thus each requiring an
independent drug development effort. Secondly, the protease must
be continuously and completely inhibited for as long as it remains
in the intoxicated neurons or a recurrence of symptoms will occur.
Thirdly, clinical trials for botulism antidote drugs will be extremely
limited and thus efficacy and toxicity from long exposure of the
drugs will remain uncertain. Therefore, it is important also to seek
alternative botulism therapies that reduce the persistence of the
BoNT proteases within neurons leading to more rapid recovery
In this study, we successfully tested the novel concept of
VHH-targeted F-box (TFB) agents to promote the accelerated,
target-specific, turnover of an intracellular protein. Specifically
we demonstrated that a VHH specific for a Botulinum
neurotoxin (BoNT) protease (Lc) and fused to the F-box
domain from TrCP will promote polyubiquitination of the Lc
in neuronal cell cytosol and accelerate its
proteasome-dependent turnover. Furthermore, we demonstrate that TFBs retain
their activity when the VHH (14 kDa) is fused to a 15 kDa
region of b-TrCP containing the F-box domain. The modular
nature of TFBs was demonstrated by replacing the ALc-specific
VHH domain with a VHH targeting the protease of another
BoNT serotype (BLc) and showing that this TFB promoted
intracellular turnover of BLc, not ALc. The potential
therapeutic application of TFBs was demonstrated by showing that
neuronal cells intoxicated by BoNT/A recovered from a
measurable symptom of intoxication (SNAP25 cleavage) at a
significantly faster rate when the intoxicated cell expressed the
appropriately targeted TFB.
This work builds on the seminal work of Zhou et al.  that
used a fusion between b-TrCP and a protein with affinity for the
normally stable retinoblastoma protein (pRB) to promote more
rapid turnover of pRB. A related strategy called Protac (Proteolysis
Targeting Chimeric Molecule) [29,30] employs small molecules
and peptides to act as a bridge between the SCF ubiquitin ligase
and protein targets leading to ubiquitination and degradation of
the target. These approaches require the identification or
availability of a targeting domain having sufficiently high affinity
and specificity to promote therapeutically useful target turnover.
Our strategy employs VHHs as the targeting domain . These
proteins derive from the VH domain of heavy-chain-only IgGs
produced by camelid animals. VHHs are small, highly stable,
single-domain binding agents that can be rapidly generated
against virtually any target by a variety of approaches 
including iterative, high-throughput methods using non-immune
libraries . The demonstration that VHHs are effective as the
targeting domains thus opens the possibility of rapidly developing
agents that target the accelerated turnover of all seven BoNT
serotypes and, indeed, most any cytosolic target within cells.
The TFB with an ALc targeting VHH fused to a full-size TrCP
proved exceedingly unstable and mostly localized to the nucleus so
an effort was made to use truncated TrCP domains that may have
improved expression and stability as well as a mostly cytosolic
localization. We hypothesized that truncation of TrCP outside of
the F-box would eliminate potential auto-ubiquitination sites and
nuclear localization signals, and would lose normal activities which
may prove harmful in a therapeutic context. We found that TFBs
with substantial TrCP truncations protected N2A cells from
BoNT/A-mediated cleavage of SNAP25 and were expressed in
the cytosol to much higher steady-state levels than TFBs with
To directly test the TFBs for the ability to promote
polyubiquitination and proteasome-mediated degradation of the
targeted BoNT proteases, neuroblastoma cells were co-transfected
with vectors driving co-expression of the TFBs and the BoNT Lc
proteases. The presence of the ALc-targeting TFB, D5-B8, was
shown to promote much enhanced polyubiquitination of ALc
compared to all controls. More importantly, co-expression of ALc
and the ALc TFB, D5-B8, led to significantly reduced steady-state
levels of ALc. Co-expression of the BLc TFB, D5D10, led to
significantly reduced levels of co-transfected BLc. The two TFBs
provided ideal negative controls for each other in these studies.
The results also validated the modular concept of the TFBs in
which the target specificity can be altered by swapping the VHH
To quantify the rate at which ALc is degraded within
neuroblastoma cells expressing TFB D5-B8 or D5-B10, stable
M17 cell lines were generated that constitutively express either the
ALc TFB, D5-B8, or TFB D5-B10. Use of these cells allows a
constant level of the TFB to be expressed throughout the
experiment and thus eliminates background from cells that do
not express TFB such as occurs with transient plasmid
transfection. Following transfection of these TFB cell lines with an
expression vector for CFP-ALc, levels of ALc were measured by
Western blot and quantified by capture ELISA. ALc turnover was
measured to be about 2.5 fold more rapid in the presence of
D5B8 as compared to D5-B10. This is a minimal estimate of the
difference in turnover rates because it doesnt account for the
continued, decreasing synthesis of ALc from the transgene which
deflates turnover estimates especially during the early time points.
The M17 cell line constitutively expressing D5-B8 was more
refractory than control cells to BoNT/A intoxication based on
cleavage of endogenous SNAP25. This indicates that much of the
ALc entering cells during intoxication becomes inhibited and/or
degraded by the presence of the TFB D5-B8. The fact that a low
level of intoxication still occurs despite the presence of D5-B8 may
indicate that ALc is partially sequestered from the cytosolic TFB
during the intoxication process and the protease gains access to the
membrane-associated SNAP25 before it can be bound by the
Finally we tested whether the presence of an appropriately
targeted TFB could accelerate the recovery of neuronal cells from
BoNT/A intoxication using SNAP25 integrity as the measure.
Since the BoNT/A protease is eliminated from intoxicated
neurons more rapidly in the presence of ALc-targeted TFB, the
intact SNAP25 should also be renewed more rapidly. Studies
demonstrated this to occur as M17 cells expressing D5-B8
recovered levels of intact SNAP25 at a significantly more rapid
rate than controls. We speculate that the TFB D5-B8 is leading to
more rapid molecular cure of the neuron through elimination of
the ALc, thereby permitting the neuron to remove cleaved
SNAP25 and renew intact SNAP25 by normal metabolism.
The studies with TrCP truncation demonstrated that much of
b-TrCP outside of the F-box domain was expendable for TFB
function. This is consistent with the dogma that it is the 50 amino
acid F-box domain that interacts with the SCF E3-ligase to recruit
associated proteins for polyubiquitination . Our results
confirm that other regions of b-TrCP are not necessary for the
recruitment of bound proteins for accelerated turnover. We also
found that the orientation of the F-box relative to the VHH did
not appear to influence the TFB function. In sum, the results
indicate that precise positioning and spacing of the recruited target
protein as it is bound to the F-box domain does not appear to
significantly influence the availability of the target protein as a
substrate for the E3-ligase.
The TFBs in this study were expressed with an amino terminal
YFP partner to permit the monitoring of expression by
fluorescence microscopy and to facilitate comparable detection
of the many TFBs tested in Western blots using anti-YFP
antibodies. The proteins also contained a small streptavidin
binding peptide to permit pull-down analysis using streptavidin
beads. While unlikely, it is possible that these fusion partners may
have some influence on the function or stability of TFBs. Once a
vehicle has been identified for the in vivo delivery of TFBs to
intoxicated neurons, further development will likely be required to
select a TFB agent having optimal therapeutic properties.
Our results indicate that TFBs have therapeutic potential as
antidotes for botulism to promote the accelerated removal of
protease from intoxicated neurons and to promote more rapid
neuronal recovery. Clearly a significant challenge to the success of
such therapy will be identifying a strategy for the delivery of TFB
biomolecules to intoxicated neurons within patients. The strategy
must deliver an effective number of molecules to a sufficient number
of intoxicated neurons to reverse paralysis. Because the amount of
protease within intoxicated neurons is very low and the TFBs may
be able to catalytically promote destruction of multiple BoNT
proteases, it is expected that the number of TFB molecules that must
be delivered for efficacy is small. The ideal delivery system should be
highly efficient and specific to neurons to minimize both the dose of
agent required and the potential for side effects from non-specific
delivery. Such a delivery system may be found in Clostridial toxins
themselves which are highly evolved for efficient delivery of
biomolecules to cells. Perhaps most promising would be BoNT
itself as atoxic versions of the holotoxin have been created and
produced in quantity  and these should be highly specific for
neurons. Also it is has been shown that a BoNT delivery vehicle
should be able to enter previously intoxicated neurons .
Furthermore, the BoNT heavy chain alone was recently shown to
be an effective vehicle for the delivery of GFP into cells . Other
Clostridial toxin-based delivery systems, such as using Clostridium
difficile toxin B (TcdB) [37,38] or Clostridial C2 toxin , may have
potential if they can be engineered to have neuronal specificity. The
small size of TFBs at less than 30 kDa makes these biomolecules
good candidates for delivery by Clostridial toxin-based vehicles as this
is smaller than the natural cargo delivered by these systems.
Materials and Methods
All protocols were approved by the Tufts University
Institutional Biosafety Committee and carried out under the CDC Select
Agent Program following all applicable federal guidelines.
Antibodies used were: mouse anti-HA antibody (Sigma); rabbit
anti-SNAP25 antibody (Sigma); goat anti-rabbit HRP antiserum
(Sigma); goat anti-mouse HRP antibody (Santa Cruz); rabbit
antiGFP antibody (Santa Cruz). Sheep anti-BoNT/A Lc antiserum
was a gift from Dr. Jean Mukherjee (Tufts University). Rabbit anti
p47 was a gift of Dr. Hemmo Meyer (ETH, Zurich). Reagents for
Western blotting, including Wash Solution and LumiGLO
Chemiluminescent Substrate, tetramethylbenzidine (TMB) were
purchased from KPL.
VHH and b-TrCP coding DNAs were amplified by PCR and
ligated into the mammalian expression vector, pcDNA3.1
(Invitrogen) fused in frame to an amino terminal yellow fluorescent
protein (YFP) coding region. The expression plasmids also contain
coding DNA for a streptavidin binding peptide (SBP)  at the
amino terminus, upstream of the YFP. The complete b-TrCP
cDNA (GenBank CAH70020) encodes 605 amino acids and
includes the 39 UTR. Truncated forms of b-TrCP all lacked the 39
UTR and contained DNA encoding the following amino acids:
D1, aa 1605; D2, aa 1379; D3, aa 1293; D4, 1233; D5, aa
175293; D6, aa 175233. ALcB8 and BLcB10 VHH coding
sequences were the same as previously reported .
The BoNT/A (subtype A1) protease (ALc) expression plasmid
contained ALc coding DNA within pcDNA3.1 fused to an amino
terminal CFP domain (CFP/ALc) as described previously .
The BoNT/B protease (BLc) expression vector was prepared
exactly as for ALc except that the ALc coding DNA was replaced
with DNA encoding BLc, amino acids 1440 from BoNT/B
holotoxin. The expression plasmid for YFP/SNAP25/CFP
indicator protein was described previously .
M17 (ATCC# CRL-2267) cells were maintained in Dulbeccos
Modified Eagle Medium (DMEM) (Gibco) containing 10% fetal
bovine serum (FBS) (Gibco). Neuro2a (ATCC# CCL-131)
(abbreviated as N2A) cells were cultured in Minimum Essential
Medium Eagle (MEME) (Gibco) plus 10% FBS. 26105 cells were
seeded into wells of a 24-well plate and maintained at 37uC.
Cell transfection and extract preparation
After 24 hrs, neuroblastoma M17 or N2A culture medium was
replaced with fresh medium before experimental treatments. Cells
at 80% confluence were used for transfection. For each well of a
24-well plate, 0.5 mg of plasmid was mixed into 50 ml of serum-free
medium. Transfection reagent FuGene HD (Roche) was added
into the plasmid mixture at a ratio of 1:3 (DNA [mg]: Fugene [ml])
and incubated at room temperature for 15 min before the
transfection mixture was applied to cells for 24 hrs. After
transfection, cells were collected following trypsin treatment and
washed once with 0.5 ml of Dulbecco s Phosphate Buffered Saline
(DPBS) for cell extract preparation. For Western blotting analysis,
total lysates were made by collecting cells in 50 ml of sample buffer
[62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol and 0.002%
bromophenol blue plus 5% beta-mercaptoethanol] and boiling for
10 min. For other applications, protein extracts were made by
collecting cells in 50 ml of lysis buffer [DPBS containing 16
protease inhibitors, 1 mg/ml BSA, 0.1% Triton-X100] and
incubated on ice for 30 min. Cell debris and protein extract were
separated by centrifugation at 13,000 rpm for 15 min at 4uC.
Streptavidin and glutathione affinity purification
25 ml of streptavidin beads (DynabeadsH M-280 Streptavidin,
Invitrogen) were washed twice with 50 ml of DPBS and once with
50 ml of lysis buffer. Washed streptavidin beads were resuspended
with 50 ml of cleared protein extract and incubated at 4uC for
16 hrs with rotation. Protein-bead complexes were washed 4 times
with 100 ml of ice cold DPBS. Bound proteins were eluted from
beads by adding 25 ml of sample buffer to the protein-bead
complex and boiling for 5 min prior to gel electrophoresis. For
glutathione affinity purification, 2 ml of glutathione magnetic
beads from MagneGSTTM Protein Purification System (Promega)
were pre-coupled with 1 mg of recombinant GST fusion proteins
by incubation at 4uC for 3 hrs. After coupling, the complex was
used to co-purify proteins having affinity to the GST proteins from
cleared cell lysates as above for Dynabeads beads. Protein eluates
in sample buffer were resolved through SDS-PAGE (415%
gradient gel, 8.666.8 cm (W6L), BioRad).
BoNT/A intoxication and transduction
M17 cells were intoxicated with BoNT/A as previously
described . Briefly, a 50 ml solution of serum-free DMEM
was prepared containing 0.75 mg of BoNT/A. FuGene HD (or
DMEM control) was then added at the ratio (BoNT [mg]: FuGene
HD [ml]) of 1:3 and the mixture was incubated at room
temperature for 15 min. The BoNT/A mixtures were then
applied to cultured cells containing 0.5 ml fresh culture medium
in a well of a 24-well plate to a BoNT/A final concentration of
10 nM. Following various incubation times, cell pellets were
collected and dissolved in 50 ml of sample buffer and boiled for
10 min prior to gel electrophoresis. To measure recovery of
endogenous, uncleaved SNAP25, 60 mm plates of M17 cells were
intoxicated with 10 nM BoNT/A for 24 hrs. Intoxicated cells
were washed twice with 5 ml of DPBS and split into 24 well plates.
Cell extracts were collected at various times post-intoxication and
processed as above.
0.5 mg of an ALc-binding VHH, JDA-D12 (GenBank accession
HQ700702), was coated onto each well of a 96 well plate at 4uC
overnight for ALc capture. Streptavidin coated plates-high
sensitivity (Thermo Scientific) were used for BLc capture. Cell
extracts prepared from transfected cells were applied onto 5%
skim milk/PBST (0.05% Tween-20) blocked plates and incubated
at 4uC overnight. Captured Lc was detected with BoNT
serotypespecific anti-Lc antisera followed by appropriate HRP-conjugated
secondary antibodies. Signals were developed by adding TMB
substrate and the absorbance was recorded at 450 nM with a
SynergyTM HT Multi-Mode Microplate Reader.
Generation of lentiviral vectors
Construction of lentiviral vectors carrying YFP-TrCP-D5/B8
and YFP-TrCP-D5/B10 coding DNA was accomplished by first
cloning each respective PCR amplified coding region into the
BamHI and XhoI sites of the transducing plasmid
pLenti6.3/V5DEST (Invitrogen). Viral particles were subsequently produced by
co-transfecting 3.5 mg of the transducing plasmid with 7.1 mg
HIV-1 gag-pol helper construct (Synaptic Research), and 2.8 mg of
VSV-G expression plasmid (Synaptic Research) onto 8090%
confluent 293FT cells (Invitrogen) cultured in 100 mm plates.
Culture medium that contained the budded viral vectors was
collected 48 hrs after transfection and cleared of cell debris by
centrifugation at 2,000 RPM for 10 minutes at 4uC (Sorvall RT
600D). The cleared viral supernatant was further concentrated by
ultracentrifugation at 25,000 RPM for 90 minutes at 4uC
(Beckman Coulter OptimaTM XL-100K). Lastly, the viral vector
pellet was soaked in 50 ml (1/200 the original volume) culture
medium overnight, resuspended, and stored at 285uC until
needed for transduction.
Production of lentivirus transduced neuronal cell lines
M17 cells were seeded onto 12-well plates the day before
transduction. On the day of transduction, lentiviral stock was
thawed and diluted to different extents into 350 ml fresh complete
medium. The original culture medium was removed from the cells
and the fresh medium containing virus was then applied to the
cells. Hexadimethrine bromide (Sigma) was added to the cells to a
final concentration of 8 mg/ml. At 6 hrs post-transduction, cells
were covered with enough complete medium for overnight
incubation. The transduction procedures were repeated on the
second day and culture medium was replaced with fresh, complete
medium containing 5 mg/ml of blasticidin to select for stably
transduced cells 24 hrs after the second transduction. Medium
with blasticidin was replaced every 34 days until only fluorescent
Figure S1 TFB ALcB8-TrCP expressed in M17 cells
inhibits BoNT/A cleavage of a SNAP25 indicator protein
and is rapidly turned over by a proteasome-mediated
process. (A) Western blots of SNAP25 indicator protein levels +/
2 BoNT/A intoxication. M17 cells were transfected with an
expression plasmid for the YFP/SNAP25/CFP fusion protein, a
SNAP25 cleavage indicator protein in which SNAP25 is flanked
by two different fluorescent proteins, YFP and CFP. The M17 cells
were co-transfected with a control plasmid (vector alone), an
expression plasmid for ALcB8-TrCP (B8-TrCP) or an expression
plasmid for ALcB8 lacking an F-box domain (B8). 24 hrs
posttransfection, cells were intoxicated by exposure to 10 nM BoNT/
A (+) or left untreated (2). Cell extracts were prepared after 24 hrs
of intoxication and the cleavage of indicator by BoNT/A was
detected by Western blot with anti-GFP antibody. The presence of
undigested indicator protein following BoNT/A exposure results
from partial intoxication of M17 cells. From prior studies, the
intoxication efficiency of these cells based on endogenous SNAP25
cleavage typically varies between 50% and 80%. Results from
three separate representative experiments (out of ten) are shown.
(B) Western blot of B8-TrCP after various times of treatment with
MG132. M17 cells were transfected with expression plasmid
ALcB8 (B8) or ALcB8-TrCP (B8-TrCP) for 24 hrs. Cells were
then treated with 10 mM of MG132 for the indicated time before
cell lysates were prepared. The expression of ALcB8-TrCP was
detected by Western blot with anti-GFP antibody. Unmodified
ALcB8-TrCP protein became much more apparent within
transfected cells following 4 or 16 hrs of exposure to MG132
and high molecular weight staining proteins accumulate. This
implies that ALcB8-TrCP TFB protein is being expressed to a
significant extent but undergoes rapid proteasome-mediated
turnover resulting in very low steady-state levels. Arrow indicates
the unmodified ALcB8-TrCP fusion protein.
Figure S2 Intracellular localization of ALcB8 TFBs
containing variable amounts of TrCP. M17 cells were
transfected with expression plasmids (as indicated) for the various
TFB proteins targeting ALc (B8-TrCP and B8-TrCP truncations)
or BLc (D5-B10) diagrammed in Figure 1A. Fluorescence
microscopy images were taken 24 hrs post-transfection to visualize
the YFP fusion partner on each TFB and the images shown are
representative of 3 separate experiments.
Figure S3 TFBs with various TrCP truncations retain
activity to protect SNAP25 indicator protein from
cleavage by BoNT/A within intoxicated M17 cells. M17
cells were co-transfected with an expression plasmid for the
SNAP25 indicator protein and a second expression plasmid for the
indicated TFB protein (diagrams in Figure 1A) or control (vector
alone). 24 hrs post transfection, cells in wells were intoxicated by
exposure to 10 nM BoNT/A (+) or left unintoxicated (2). Cell
extracts were prepared after 24 hrs of intoxication and the extent
of cleavage of the indicator by BoNT/A was assessed by Western
blots with anti-GFP antibody and the results shown are
representative of 3 separate experiments.
Figure S4 TFB D5-BLcB10 expressed in N2A cells binds
to BoNT/B Lc target. A. TFB D5-BLcB10 (D5-B10) binds to
co-expressed GST-BLc in N2A cells based on GST pull-down.
Glutathione-transferase (GST) fused to BoNT/B Lc (GST-BLc),
or GST (control), each complexed with glutathione magnetic
beads, was added to TFB D5-B10 transfected N2A cell extract and
the GST proteins were recovered by glutathione affinity. D5-B10
was detected by anti-GFP antibody and shown to be present
following GST pull-down of the BLc. B. D5-B10 binds to
coexpressed GST-BLc in N2A cells based on streptavidin pull-down.
N2A cells were co-transfected with expression plasmids for
GFPBLc and for TFB D5-B10 (fused to streptavidin binding peptide
and YFP). The D5-B10 was purified by streptavidin affinity and
the pull-down fraction was analyzed for co-purified GFP-BLc by
Western blot using anti-GFP antibody. An equivalent aliquot of
the unpurified cell extract (input) was also included on the Western
blot. Data shown are representative of 3 separate experiments.
Figure S5 BoNT Lc targeted TFBs reduce the
steadystate level of the targeted Lc in neuroblastoma cells
expressing both ALc and BLc simultaneously. N2A cells
were co-transfected with expression vectors for both CFP-ALc and
CFP-BLc along with another expression plasmid for either the TFB
D5-B8 or D5-B10 as indicated. 24 hrs post-transfection, cell extracts
were prepared and resolved by SDS-PAGE. CFP-Lc and SNAP25
expression levels were detected by Western blotting using BoNT
serotype-specific anti-Lc antisera (ALc or BLc) or anti-SNAP25
antibody. The data shown is representative of 2 separate experiments.
Figure S6 ALc turnover is accelerated by co-expression
with TFB D5-B8. Expression plasmid for CFP-ALc was
transfected into cells stably expressing D5-B8 (ALc TFB) or the
control, D5-B10 (BLc TFB), as indicated. Cell lysates were
prepared at the indicated time points and resolved by SDS-PAGE.
The expression level of CFP-ALc, TFB and SNAP25 were
assessed by Western blotting using anti-ALc Ab, anti-GFP Ab or
anti-SNAP25 Ab for detection and the data shown are
representative of 3 separate experiments.
Figure S7 TFB-mediated acceleration of BoNT Lc
turnover is proteasome-dependent. N2A cells were
cotransfected with expression plasmids for CFP-ALc or CFP-BLc
and expression plasmids for TFBs D5-B8 or D5-B10 as indicated.
24 hrs post-transfection, cells were treated with either 10 mM
MG132 or a DMSO control. 4 hrs later, cell lysates were prepared
and resolved by SDS-PAGE. CFP-Lc expression levels were
detected by Western blotting using BoNT serotype-specific anti-Lc
antisera and the data shown is representative of 4 separate
Figure S8 TFB targeting ALc protects cells from
cleavage of endogenous SNAP25 following BoNT/A
intoxication. M17 control cells or M17 cells stably expressing ALc TFB
(D5-B8) or BLc TFB (D5-B10) were intoxicated with 10 nM
BoNT/A for 5 hrs. Cell lysates were prepared and resolved by
SDS-PAGE. The level of SNAP25 cleavage was detected by
Western blotting using anti-SNAP25 Ab (A) and the % cleavage
was quantified by scanning densitometry (B). Data are presented as
the averages 6 standard deviation calculated from three
experiments and compared by ANOVA. The differences between
ALc TFB line and BLc TFB line, and between ALc TFB line and
M17, are highly significant (p,0.005).
Figure S9 ALc TFB (D5-B8) expression promotes
accelerated recovery of intact endogenous SNAP25 following
BoNT/A intoxication. Control M17 cells or M17 cells stably
expressing ALc TFB D5-B8 or BLc TFB D5-B10 were exposed to
10 nM of BoNT/A for 24 hrs. Cell lysates were prepared at times
indicated post-intoxication and resolved by SDS-PAGE. SNAP25
was detected by Western blot using anti-SNAP25 Ab and the data
shown are representative of ten separate experiments.
We are grateful to Dr. Jean Mukherjee for providing antisera and mAbs
recognizing BoNT proteases, Dr. Hemmo Meyer for providing the
antip47 sera, Dr. Yung-Nien Chang for preparing and providing the
lentiviruses and to Dr. Randall Kincaid for providing the GST fusion
proteins to ALc and BLc and the indicator protein expression vector. We
thank Dr. Saul Tzipori for his helpful advice and consistent support of this
project, and Drs. Ira Herman, Hanping Feng, Jong-Beak Park and Patrick
Skelly for helpful discussions.
Conceived and designed the experiments: CS CK. Performed the
experiments: CK. Analyzed the data: CK GO CS. Contributed
reagents/materials/analysis tools: GO. Wrote the paper: CS CK.
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